Aedes aegypti, a vector of numerous human pathogens. They can be used to explore multiple biological questions related to population size, dispersal, and other interactions related to the spread of mosquito-borne diseases. To ensure marking practical aspects and that these powders do not interfere with mosquito natural behaviors, differentiation of multiple colors applied externally in the same mosquito, their impact on mating, and their transference between individuals after copulation and same-sex interactions were studied. Multiple color differentiation was possible, except for green–yellow combination. No important effect of powder marking was found on any of the mosquito mating phases: coupling , copulation , and insemination . After copulation with a powder-treated mate, >80% of females and all males had powder in genitalia, legs, and wingtips. The transfer of powder, between same-sex individuals, occurred only in males. In general, fluorescent powders had a little observable effect on Ae. aegypti mating, suggesting that these markers do not alter this important vector life-history trait and are a useful and viable tool for mosquito studies.Fluorescent powders are one of the most used materials for externally marking mosquitoes, such as Aedes aegypti adults to evaluate the differentiation of multiple fluorescent powder colors applied externally in the same female mosquito, their effect on coupling time, copulation time, insemination success, mate choice, and the extent of transference of powders between marked and unmarked individuals, either during copulation or same-sex interactions. Marking with multiple powders was evaluated after applying different powders in the same female at different times and combinations. The comparative effect of powders on mating was explored using different cross-combinations of marked/unmarked couples. Transference of powders between marked/unmarked individuals after copulation was checked in mated individuals, and between same-sex interactions by allowing them to interact under crowded and uncrowded conditions. Identification of the colors included in multiple markings in the same individual was possible when exploring almost all combinations (exception: green–yellow). No important effect of powder marking between cross-combinations was found on coupling time  37.6–49.6 min), copulation time , insemination success, nor their mate choice. Transferred powder after copulation activity, concentrated in genitalia, legs, and the tip of wings, occurred in >80% of females and 100% of males. Powder transference in legs and genitalia, between same-sex individuals, occurred only in males (ranged between 23–35%) under both density conditions. The lack of important effects of these powders on the studied aspects of Ae. aegypti provides information about their usefulness and limitations, which should be recognized for future applications and to avoid bias.Five different fluorescent powders  were tested on  Aedes aegypti (Linnaeus) females, have also revealed their low impact on survival of different age cohorts, female blood-feeding success, tethered flight velocity, and their recapture in baited traps [A wide variety of methods and techniques for marking mosquitoes have been developed, many of which have been used to explore multiple biological questions related to population size and dynamics, dispersal, feeding behavior, trophic-level interactions, and other ecological interactions ,2. Amonged traps ,5. OtherDrosophila flies [Diabrotica virgifera [Multiple marking of the same individual mosquito during different moments of their life span or activity periods, expand the practical uses of the fluorescent powders as marking tools. This technique allows the time determination of successive recaptures, that inserted in different types of studies, can be used for exploring population size, dispersal, survival, age structure composition, feeding behavior, and overcome limitations when working in low-density habitats . Markingla flies  or the cirgifera , have usAe. aegypti mating has not been studied in detail. Experiments exploring the effects of other variables on mating characteristics of Ae. aegypti males, such as male mating competitiveness [Metarhizium anisopliae infection on mating activity [To our knowledge, the effects of fluorescent powder marking on tiveness  or the eactivity , have usAedes aegypti mating takes place naturally mainly in aerial swarms near the host [the host ,12,13 orthe host ,15, whenthe host ,17. The the host . Duratiothe host , their fthe host . Reportethe host ,20,21. Fthe host ,22. In cthe host ,24.Psorophora mosquitoes heavily marked with fluorescent powder can transfer it to 1–3% of unmarked individuals in a confined space [Culicoides biting midges and unmarked controls nor to the environment in which the marked individuals were held for a 24 h period [Drosophila pseudoobscura dusted nonvirgin males and females; some genital transfer if nonvirgin individuals were allowed to clean themselves before mixing them under moderately crowded conditions; and no transfer during mating of virgin adults [Anopheles arabiensis found that males are capable of transferring the mark, only detectable when examined under a stereoscope, to both males and females and that this transference increased over time [Culex quinquefasciatus marked males and unmarked females [Ae. aegypti, as a tool for distinguishing between treatment and control groups [Another experimental aspect of markings that can generate biased results is powder transference. Markers can be transferred from intentionally marked individuals to unmarked individuals when mosquitos interact inside collecting devices or in field conditions. This has been considered unlikely when aspirating mosquitoes in groups, and not observed when collecting mosquitoes with CDC (Centers for Disease Control and Prevention) light traps, or when aspirating mosquitoes individually or using a battery-powered aspirator . Other eed space , but no h period . Powder n adults . Studiesver time , but no  females . Transfel groups .® ECO Series [Ae. aegypti adults to evaluate the differentiation of multiple fluorescent powder colors applied topically in the same female mosquito, their effect on coupling time, copulation time, insemination success, and mate choice, along with their transference between marked and unmarked individuals, either during copulation or same-sex interactions. The selection of these powders is related to their performance in previous experiments [From a practical perspective, the evaluation of multiple marking and powder transference between individuals is crucial for validating their use and recognizing their limitations or potential bias. From a biological point of view, understanding their impact on the mosquito mating process and their mate choice is essential, since the conclusions of any study using this tool, should be aware of their potential impact on male and female mating interactions. In this study, five different Day-GloO Series  fluoresceriments ,5.® ECO Series [Ae. aegypti adults to evaluate their differentiation in the same individual after being marked with more than one powder, their effect on the mosquito mating process and mate choice, and their transference between marked and unmarked individuals. The experimental codes of the fluorescent powders (color-powder identification) used were: DG1 (fire orange—ECO 14), DG2 (Saturn yellow—ECO 17), DG3 , DG4 (horizon blue—ECO 19), and DG5 (ultra violet—ECO 20).Five different water-insoluble fluorescent powders, with a specific gravity of 1.2 and a mean particle size of 4.5 µm, of the brand Day-GloO Series  were tesAe. aegypti adults  were performed as previously described [Protocols for rearing and marking escribed . Brieflyescribed ,31. Presescribed .Individuals from two control groups were used. One control group had females that were not handled or exposed to any powder (control colony: CC), and the other sham control group had females exposed to the marking technique but without using any powder (control application method: CAM). Different combinations of unmarked female (UF), marked female (MF), unmarked male (UM), and marked male (MM) were used.To investigate the influence of the application time and the recognition of two different powders in the same female, groups of 10 females were exposed to one fluorescent powder at time 0 h, and then independently to a second powder at four twelve-hour intervals . Three replicates of twenty color combinations, exploring different color orders and time of application for all 5 powders, were performed. After the application of the second powder, females were collected, frozen, and inspected, in order to establish if both powders can be recognized separately in the same female and to give them a score.t = 24 h, a third powder at t = 48 h, a fourth powder at t = 72 h, and a fifth powder at t = 96 h. In this approach, 35 females were used per combination, and one hour after each application, 5 randomly selected females were collected, frozen, and analyzed in order to establish if sets of powders can be separately recognized in the same female. The order of colors in the two combinations was established by using the color wheel: one combination was in a clockwise direction (yellow–orange–violet–blue and green), and the other in the counter-clockwise direction.To study more than two powders in the same female, three replicates of the two combinations were explored. Females were exposed to one powder at time 0 h, were re-exposed to a second powder at Ae. aegypti mating process , and mate choice. The same rearing protocol described previously [Different cross-combinations of marked/unmarked males and females were used to analyze the effect of fluorescent powders on eviously  was folleviously .Ae. aegypti along the cross-combinations was explored by exposing one virgin male with up to 3 consecutive virgin females [The comparative effect of fluorescent powders on the mating process of  females ,20. Adul females . Twenty  females ,20. The  females ,20, but  females . The mov females , and a fTo investigate if the presence or absence of fluorescent powders in females affects the male sexual mate choice, 5 unmarked virgin males were placed simultaneously with 10 marked and 10 unmarked virgin females inside a translucent polyester mesh cage (W32.5 × D32.5 × H32.5 cm) for 24 h. After this period, males were removed, and females were left inside the cage for 24 h more . Then, tMosquitoes used in both experiments were stored into 1.5 mL Eppendorf tubes and frozen for wing length measurements. The wings were removed under a stereo microscope and then mounted on microscope slides using double-sided tape. Wing digital images and measures, from the auxiliary incision to the apical margin excluding the fringe , were taThe transference of powders between individuals during copulation was studied using the marked/unmarked males and females from the different cross-combinations of the mating process and mate choice experiments. Their powder score, complemented with the description of the part of the mosquito body where it was observed, was recorded, and analyzed.The transference of fluorescent powders between same-sex individuals was tested by allowing them to interact under two density conditions: crowded and not crowded. Three replicates of four different combinations of 3–4 day old marked (marked 24 h before the interaction) and unmarked females and males , were placed inside paperboard cages with mesh tops (H10 × top D10 × bottom D7 cm) in equal ratios, with a total number of 20 (not crowded conditions: NC) and 100 individuals (crowded conditions: C), respectively. Twenty-four hours after exposure, all specimens were frozen, individually examined, and scored. The same selection of powders was used as already described in the mating process experiment.p-values using the Bonferroni method [Statistical analyses were performed in IBM SPSS Statistics . A two-si method . For allFor the mating process experiment, a time-to-event analysis  was used for analyzing the coupling time or length of time until the occurrence of copulation events after each female was introduced to the male. One hundred minutes was used as the endpoint time of observation, the different cross-combinations as the factor of comparison, and age, wing size, and female sequential order of presentation to the male as covariates. Information on mosquito couples that had not copulated by the end of the observation period was censored. One-way analysis of variance (ANOVA) was conducted to compare, both mosquito females and males, wing size (mm) among cross-combinations. Using only the information of males that copulated with three females (N:25), a profile analysis, with one within-subjects factor  and one between-subjects factor  was conducted to examine if the copulation time (s) of each male, across all cross-combinations, varied after the successive copula with three sequentially presented females. A linear least-squares regression was used to predict the effect of the different cross-combinations on the number of filled spermathecae in inseminated females. The same type of regression was also used to assess if the score obtained in the females that had transferred powder after copulation was affected by the cross-combinations 1 (MM-MF) and 2 (MM-UF), age and size of the females and males, and the female order in which they were presented to the same male  .The distinction of more than two colors in the same female, required the use of magnification (stereo microscope). Determination of color when a single color is used can be done accurately and easily with the naked eye or using UV light.To analyze the effect of application time and the recognition of two different powders in the same marked female, 600 total females were analyzed. The combinations of yellow–orange, green–orange, and blue–orange were recognizable and all tested females had both powders in all their five body parts (score of five), regardless of the order of application and time elapsed between applications. The easiest combination to distinguish was when using orange and yellow together. Combinations of blue–yellow and blue–green showed similar results as the latter, with the difference that when the powder blue was applied second, its visualization was more difficult and only aided by the clustering of the powder in a body region. Regarding all the previous combinations, the pigment that was placed first usually remained in greater coverage and therefore was more visible than the second pigment. Irrespective of the application time or order, the combination of green–yellow was not distinguishable. The use of violet was also hard to identify and differentiate from the mosquito scales, which seem violet when observed under the UV light. Blue and violet powders look duller when compared to the other brighter colored powders.To study more than two powders in the same female, 150 females total, grouped in two combinations, were analyzed. Recognition of each powder, after multiple powders application, was similar to what was observed when two of them were applied. Combinations of orange–yellow and green–blue (at 24 h) were recognizable, but the third powder (violet) at 48 h was not visible. The application of a fourth powder was feasible, but easier in the case of orange than in the case of blue. The application of the fifth powder (green or yellow) presented the same difficulty previously expressed, that is that the combination yellow–green, regardless of the order of application, is not visually distinguishable. All the females marked on multiple occasions with powders that were distinguishable, presented a score of five, irrespective of the application time or order.Ae. aegypti mating process . Thirty-seven males did not copulate with any female by the end of the exposure time, 19 with one, 19 with two, and 25 with three females. Since the consecutive female (second or third) was presented to the same male after he copulated with the previous one, not all 300 available females were used, and only 207 were exposed to a male. Of this number, 132 females did copulate with the male, and 75 did not copulate by the end of the observation time. The frequency of copulation events was similar among mosquitoes of different ages (from 5 to 18 days old). Mean wing size of males (2.06 ± 0.06 mm)  and females (2.74 ± 0.15 mm)  were not significant different between cross-combinations.One hundred males were used for the analysis of the effect of fluorescent powders on the 2 = 2.36, p = 0.666, N = 207) (Coupling time (min) or length of time until the occurrence of copulation events after each female was introduced to the male was no different among cross-combinations (XN = 207) . Overall8,38 =2.075, p = 0.063). The test of between-subject effects or differences in the means between cross-combinations groups showed that there was not a significant difference in copulation time between the cross-combinations groups  . The sphericity assumption, an assumption of equal variances in the differences between all possible pairs of within-subject conditions, was not violated .The presence or absence of powder in the different cross-combinations and the order of female presentation had no observable effect on copulation time . Overallp = 0.150), nor was a predictor of the number of filled spermathecae in inseminated females . Of 11 females that did not copulate, five were presented first or second in sequence to a male that was able to copulate and inseminate with the next female.Insemination success was high across all treatments. Of the 132 females that copulated , 91% were successfully inseminated. Almost 70% of the inseminated females had three spermathecae filled, and none of them had one . Of the 1,113 = 0.21, p = 0.885).To investigate if the presence or absence of fluorescent powders in females affects male sexual mate choice, a total of 30 unmarked virgin males (wing size 2.07 ± 0.06 mm) and 115 virgin females (wing size 2.71 ± 0.09 mm) were used. No significant variation in the female mean wing size between marked and unmarked females was detected by the ANOVA . No significant variation was found either in the proportions of inseminated or not inseminated females in marked or unmarked females (p = 0.162), and the proportions of inseminated marked or unmarked females with one, two, or three filled spermathecae (p = 0.128). More than 50% of the inseminated females had three spermathecae filled, and in all cases, the medial spermatheca was the first to be filled  were not significantly different . The least-squares regression (R2 = 0.59) to predict the female scores obtained due to copulation interaction revealed that only cross-combination  and female size  were significant predictors of the least squares mean (LSM) female powder score . The relationship between the wing size and score was negative (t Ratio = −2.20), suggesting that larger females presented lower powder scores.For the mating process experiment, proportions of copulated females with transferred powder were not significantly different between combinations 1.MM-MF (88%) and 2.MM-UF (100%) , and wings (score three). The rest of the score combinations included females with powder also in the proboscis and thorax. Almost all the males used in the mate choice experiment showed the powder in their genitalia, legs, and the tip of the wings and proboscis .p = 0.49) or among combinations MM-MM/MM-UM  (p = 0.521). An average of 74% of the males with transferred powder, exhibited the powder in legs and genitalia , while the rest of them in the legs, genitalia, and wings.The transference of fluorescent powders between same-sex individuals happened in a similar way among both density conditions and combinations, but different amongst sexes. In the case of females, no evidence of transference of powder was observed, regardless of the combination or the density condition. In the case of males, transference was observed, but the proportions of males with transferred powder were not significantly different among density conditions C: 25%—NC: 28% ( > 0.05) . ResultsDifferent descriptive aspects about the use of fluorescent powders in the multiple marking of mosquitoes are presented. This knowledge can help in decision making and defining their functionality when used as tools in different experimental settings. The limitations of using florescent powders can vary, depending on the detection system being used. For example, when human sight is the method for discriminating different colored powders, it is important to evaluate practical aspects of the experiment before committing to detailed protocols. Differences between experimental findings using powders can be linked to the use of different types of powders, powder concentration, method of application, and marked adult grooming behavior. Additionally, there are consistent reports of distinguishable combinations of orange with yellow, green, or blue and indistinguishable combinations of yellow–green ,8. The hDrosophila adults, we found that the order of application had an effect in determining which powder was most easily detected, but not defining whether the two-powder combination was distinguishable from either powder alone [In contrast to observations made in er alone . It is per alone . This ouer alone  with theer alone . This aser alone , and autAe. aegypti mating, a high percentage of copulation events, no difference among combinations in the coupling time, copulation time, insemination success nor mate choice, suggest low effects of these powders on the mentioned traits. Lack of studies evaluating this mating interaction in marked Ae. aegypti mosquitoes prevent us from establishing comparisons under similar conditions. Records of previous studies that have also used fluorescent powders, in both controls and treatments, for exploring Ae. aegypti copulation traits have demonstrated successful copulation and insemination in the control marked individuals [Regarding the impact of fluorescent powders in ividuals ,10.Ae. aegypti natural mating state, ready to copulate when the proper stimulus is supplied [Occurrence of copulation events scattered throughout couples of different ages and after successive copula, in both marked and unmarked individuals, is in accordance with a life-time supplied . Althougsupplied ,12,16,17Ae. aegypti unmarked males. In the mentioned study, the total time for single males to attempt or to successfully copulate ranged between 50 to 150 min, when using a single control marked male with five virgin female mosquitoes in successive copulation [Ae. aegypti males . Our rean of 16  in a secAe. aegypti mosquitoes of different ages [Ae. aegypti male capacity of inseminating 4–6 females consecutively [Ae. aegypti to copulate freely [A high percentage of insemination success in all marked or unmarked copulated females, as when analyzed in first, second, or third in the sequence, is consistent with reports of insemination rates in laboratory conditions of 74–100% using ent ages ,34,43 ancutively ,23,24. Le freely ,34, but e freely ,44. All e freely ,20,34,44e freely . In othee freely  in low cAe. aegypti electroretinographs, since as it is already reported, females show a maxima spectral sensitivity in the ultraviolet and green wavelength ranges [The lack of male preference to a marked or unmarked female in the mate choice experiment, as well as no difference in the percentages of inseminated females in either group, suggests that the presence of fluorescent powders was not a barrier for mating decisions and ability to copulate and inseminate females. Future experiments can explore the effect of marked individuals in unmarked h ranges .Ae. aegypti mating, it is important to specify that the natural mating behavior of Ae. aegypti is rarely simulated in the laboratory and therefore, the conclusions from these experiments are condition dependent, i.e., apply only to circumstances similar to these laboratory protocols. Additionally, the female reproductive success after mating was not determined in marked females or unmarked females that copulated with a marked male.As part of the limitations of the study regarding the impact of fluorescent powders in Wolbachia-infected Ae. aegypti [Ae. aegypti with wild mosquitoes [In relation to the powder transference, we found evidence for asymmetric transference of fluorescent powders between same-sex interactions among mosquito sexes. The observation of transference solely between males, together with the lack of density effects, suggests that the mechanism is linked to male behavior. Observations of virgin males allowed us to record male–male interactions similar to male–female copulation contacts . This be aegypti  and RIDLsquitoes .Ae. aegypti mating behavior, that involves the male legs seizing specific parts of the female appendages during the coupling, and then the clasping of the tip of the female abdomen with his terminalia to insert his aedeagus into the genital chamber during the copulation [Powder transference in copulation was observed in the majority of females exposed to marked males. The presence of the powder predominantly in the legs and genitalia, and then in wings, is related to the pulation . Not allpulation . These lpulation . A highepulation , and in pulation . It is pAe. aegypti [An. arabiensis [Psorophora mosquitoes [Ae. aegypti [Psorophora mosquitoes [Culicoides [Drosophila [Our findings are consistent with those found using  aegypti , An. araabiensis , and Psosquitoes , where t aegypti , a lowersquitoes , or the licoides  and in Dosophila  during mosophila . AlthougAe. aegypti coupling time, copulation time, insemination success, and mate choice, as well as the possibility of using them in studies that include multi-marking, make these powders a useful tool available for studies where those considerations are important. Recognition and attention of possible powder transference are necessary to avoid unnecessary bias. Future practical applications of these powders can be related to a trap development, in which the powder is administered automatically, and a sensor is able to record each marked individual that enters and exits the trap, eliminating all human handling. Additionally, due to the lack of effect of these powders on the several mosquito traits analyzed here, as well in past studies [The lack of evidence that the tested fluorescent powders affect  studies ,5, other studies .


Among the vast variety of plant-derived phytochemicals, the group of carotenoids has continuously been investigated in order to optimize their potential application in the area of dietary intervention related to chronic diseases. One organ that has been especially targeted in many of these studies and clinical trials is the human prostate. Without doubt, carotenoids (and their endogenous derivatives—retinoids and apo-carotenoids) are involved in a plethora of intra- and intercellular signaling, cell growth, and differentiation of prostate tissue. Due to the accumulation of new data on the role of different carotenoids, such as lycopene (LYC) and β-carotene (BC), in prostatic physiology and pathology, the present review aimed to cover the past ten years of research in this regard. Data from experimental studies are presented in the first part of the review, while epidemiological studies are disclosed in this second part. The objective of this compilation was to emphasize the present state of knowledge about the most potent molecular targets of carotenoids, as well as to propose promising carotenoid agents for the prevention and possible treatment of prostatic diseases. In the first part of the review , we coveThis second part of the review is focused on the latest research in the field of associations of carotenoid intake and/or circulatory concentrations related to the incidence of malignancy of the most important pathology of prostate—prostate cancer (PC). Today, PC is the most abundant cancer in men worldwide . In 2018Presently, the average age of diagnosis of PC is 66 years and the general belief is that below the age of 50 years its incidence dramatically drops, with only 1 in 350 younger males being affected by the disease [Since the previous decade, it has been hypothesized that carotenoids (particularly lycopene (LYC)) might exert a positive influence on the clinical outcomes of PC prophylaxis and treatment. Although LYC has been shown to improve the incidence and prognosis in humans and animals regarding gastric, breast, pancreatic, colon, and renal cancer, there have also been contrasting results reported in this area of research. Additionally, there is a scarcity of high quality observational and interventional studies performed in order to unequivocally establish the role of LYC in cancer disease ,6. In teIn fact, it is reported to possess the strongest antioxidant properties among all the carotenoids, which is accomplished by both its ability to directly scavenge reactive oxygen species (ROS) and to upregulate the antioxidant response elements (ARE) . TherefoThe main aspects of carotenoids’ physiology and molecular mechanisms of action have been summarized graphically .The fundamental issue that has hampered previous attempts to define the precise role of various factors related to PC progression (not only carotenoids) is the fact that the exact biology of PC itself is still under investigation. In 2015, the team setting up the The Cancer Genome Atlas proposed a new division of PC into seven molecular subtypes, differing in their androgen receptor (AR) activity in a subtype-specific manner . In thisHere, we provide a brief description of the methodology. A full description of the methodology was given in part I .We have investigated electronic databases  in order to find relevant studies for our literature review. Studies considered included both intervention trials and observational trials determining the association between any carotenoids and prostate complications. Investigated populations included elderly men with prostate abnormalities (predominantly PC). For interventional studies, the independent variable was the amount of supplemented carotenoids or their serum concentration after intervention. Outcomes were assessed as the presence of disease—for PC, measured mainly by screening tests, PSA, and digital rectal examination (DRE). We decided to extract data from papers published between the 1st of January 2009 and the 15th of November 2020.All received results were manually checked by two authors. The main eligibility criteria were: studies investigating the impact of any carotenoid on aspects of prostate physiology and/or pathology.Two authors were chosen to independently find studies, which met the inclusion criteria. If there were any conflicting situations, a third author was engaged for a final decision.An analysis of epidemiological studies was done following the Meta-analysis of Observational Studies in Epidemiology (MOOSE) standard . HoweverThe flow chart summarizing the process of data extraction is presented as We identified 23 research articles referring directly to the influence of carotenoids on PC occurrence. All but one were performed with human subjects. The predominant investigated carotenoids were LYC (n = 14) and β-carotene (BC) (n = 13). In addition, articles contained results in relation to retinol (or retinyl esters), α-carotene, β-cryptoxanthin, zeaxanthin/lutein, phytoene, and phytofluene. Regarding the type of study and its design, there were 2 controlled clinical trials, 6 cohort studies, 11 case-control studies, and 3 cross-sectional studies. The largest one was the Health Professionals Follow-up Study, which presented 5728 PC cases. Basic information about each study  is shown in Due to the qualitative nature of the analysis, the assessment of risk of bias was done adhering to the GRADE-CERQual. Following this step, we focused on methodological limitations, coherence, adequacy of data, and relevance of included studies .Number of cases—eight studies included less than 100 cases of PC ,20,21,22Nationality of participants—the nationalities that have been most frequently investigated are: American (n = 6), Finnish (n = 5), and Italian (n = 2). There were also Australian, Japanese, and Vietnamese populations. Four studies included multiethnic groups of participants. The prevalence of PC strongly differed, depending on the geographic region. According to the Global Cancer Statistics, in northern America, PC incidence is 73.7 , 85.7 in northern Europe, 60.7 in southern Europe, 86.4 in Australia, 13.9 in eastern Asia, 12.7 in southeastern Asia, and 64.1 in southern Africa  . It is pEvaluation of outcome—this is probably the most confusing factor explaining the high level of inconsistency among results of different studies. First, none of the prospective studies could guarantee that the outcome—PC development—was not present at the beginning of study. For example, the two included controlled clinical trials ,33 extraIn turn, there is one important example of how post-research evaluation is likewise important, as presented by Kristal et al. . During 4.Evaluation of carotenoids—there were three main methods used for calculating a relationship between PC incidence and carotenoids: (a) patients were given supplements containing a known quantity of carotenoids ,23; (b) 5.Studies that tested supplementation of carotenoids did not evaluate carotenoid dietary intake in any way. For example, in the CARET study, the only limitation for a participant was not to use dietary supplements containing >5500 IU/day retinol or any BC . However6.Measurement of serum concentrations of carotenoids was the predominant method in the investigated studies (11 of 22). However, the course of the procedure of taking blood samples varied strongly among different studies. Only in the KIHD study was the procedure precisely described, which included: (i) definition of times for taking blood samples (between 8:00 AM and 10:00), (ii) definition of the site of sampling , (iii) presampling recommendations for patients , (iv) definition of the technique for taking blood samples (blood sample was taken without tourniquet and after 30 min of resting in supine position) ,16. NoneIndeed, serum concentrations of carotenoids may differ strongly depending on the time of sampling. Among the analyzed studies, in four of them, more than one single sampling of blood was carried out ,26,33,34The least heterogeneity among studies was observed regarding the methodology of laboratory analyses. The single and only method used was high performance liquid chromatography (HPLC), as this is the routine technique for carotenoid detection and quantification, though also HPLC coupled with mass spectrometry (LC-MS) is becoming more common ,41,42, e7.Studies that investigated the effects of dietary carotenoids used a slightly different way to assess their results. In the Health Professionals Follow-up Study (HPFS) study, data collection was conducted through a standardized 131-food-item semi-quantitative Food Frequency Questionnaire (FFQ). Respondents were obliged to fill out the questionnaire a total of six times to eliminate the influence of dietary habit changes during the twenty-year-long study period. Questions about frequency and type of consumed food were asked. This is an often used technique for estimating the daily intake of carotenoid-rich food items. The content of each carotenoid was then calculated by means of data from the US Department of Agriculture (USDA) food composition database ,22, whic8.The overall quality of studies was high or moderate. Controlled clinical trials ,23 proviAll cohort studies ,30,35,37Among case-control studies, some received lower scores according to NOS scale because: the exclusion of PC in the control group was not done, the control population was selected from a specific group , the exposure assessment was poor  or they were lacking information about blinding to case/control status of interviewer, or the information about nonresponse rate was not provided. Therefore, four case-control studies ,24,27,32No major concerns regarding the quality of cross-sectional studies were raised—whether it must be taken into consideration, that their results could be only supportive in any sort of concluding about the relationship between carotenoids and PC incidence.In the following, we discuss results of our findings separately for LYC, BC, and retinol. This approach was due to the different biology and absorption distribution, metabolism, and excretion (ADME) of each carotenoid. Indeed, their effects were reported to be either different or even antagonistic.Before focusing on the human studies, we will first discuss the one animal study that was dedicated to analyzing the risk of PC. It drew some conclusions that appear to be important for a better understanding of results and restrictions of the human studies. The study was conducted by Conlon et al. in 2015 and investigated whether supplementing, in a TRAMP mice model, with 10% tomato powder (TP), would influence the incidence of PC. The duration of the intervention was eight weeks. In this study, the effects of LYC and BC were examined. The only results that reached significance were inconsistent with the time trend—LYC was shown to reduce the risk of adenocarcinoma after sixteen weeks, but not in groups of mice after twelve or twenty weeks. Another finding of this research was more indicative. The tissue of distribution of carotenoids differed strongly and depended on serum carotenoid concentrations in a nonspecified way. For LYC, the liver concentration was almost linearly related to serum concentration. For gonadal adipose tissue, there was a trend found. Finally, in testes and anterior prostate , LYC concentrations were constant regardless of the changes in serum concentrations. BC was not measured in tissues due to its low levels (below the limit of detection—0.5 nmol/g) , which ip < 0.0007) [Among the 13 human studies investigating LYC . However, considering the intake of LYC derived only from canned or cooked tomatoes , a strong protective effect against PC was revealed , and distinctly so for the aggressive disease in the age-adjusted model , but not significantly in the multiadjusted model). The study introduced the interesting idea that the processing of LYC-containing products might in fact enhance its capability to limit the growth of PC. The authors’ suggestion was that the observed effect stems from an increased bioavailability of a heated or oil-treated LYC-rich matrix  [The newest report touching on the issue of tomato intake  is AHS-2, covering the cohort of 27,934 men and 1226 PC cases from America and Canada, who were followed-up with over 7.9 years. The study was based upon FFQ questionnaires used to estimate daily LYC intake (with particular attendance referred to tomatoes and tomato-based products). The mean LYC intake was 6.1 mg per day in men without PC and 5.8 mg in those who developed PC of any severity (ns form) .Results of the HPFS study showed that dietary LYC was associated with a protection from developing PC. Results were adjusted for almost all confounding factors that could be interfering —only alcohol intake was not included. The analyzed groups were the largest ones of those included in the present review. Also, the dietary questionnaires were taken six times, thus the changes in nutritional habits were closely monitored. The main concern was that all participants were healthcare professionals and, therefore, might not be a representative group of the whole community. Analysis for TMPRSS2: ERG associations was naturally limited only to subjects who underwent a radical prostatectomy, as this specific translocation is acquired in prostatic tissue, while dysplastic changes are further developing. It is worth noting that a reverse association with PC was even stronger for tomato sauce than for LYC alone, as shown by Graff et al. . As the Out of the three retrospective studies, one found a positive correlation between LYC and PC, however it still referred only to one specific type of PC—detected despite any indication by biopsy . It was based on results from the Prostate Cancer Prevention Trial (PCPT), which originally tested the efficacy of 5 mg per day of finasteride, an 5-α-reductase type 2 inhibitor, for PC prevention. The study assessed LYC serum concentrations but also included prostate biopsies collected every year, which enabled researchers to avoid bias due to the PSA-dependent underdiagnosis of the disease. Almost 90% of cases were white Americans, thus the ethnic structure was similar to that of the HPFS study. LYC concentrations were stratified by quartiles . In the placebo arm, the only statistically significant result was a higher risk of PC not found by PSA screening in the fourth quartile—HR was 1.42 . Interestingly, in the finasteride arm, the frequency of PC detected due to the raised PSA was lowered; HR was 0.57 . Simultaneously, if already diagnosed for cause, the disease was more aggressive and presented a higher grade or stage. There were about 50% more cases of PC in Gleason stages 7–10 in the for cause group than in the not for cause group [In a somewhat disturbing conclusion, it was thought that the protective effect of LYC might merely be due to lowering the sensitivity of the PC screening instead. Not-for-cause-detected tumors in PCPT comprised 46.8% of the total cases. In the highest quartiles of LYC, the quotient of risks of not for cause/for cause were increasing strongly. It cannot be compared to any other study, as prostate biopsies have not been used commonly in studies. It is possible that LYC could, in fact, impair detection of a low-grade disease, which would not turn out to be harmful even in long-term perspective.Another case-control study was carried out in Vietnam by van Hoang et al. in 2013-2015 . Within Despite the results being generally in agreement with the findings of the HPFS, there are some differences that impede the comparison of their conclusions. First, the Vietnamese study was retrospective and, in addition, data were extracted from the population of a single city. There were also significant changes between the questionnaires  compared to the HPSF. Interestingly, this study put into a question the assumption that in Asiatic populations the effect of tomatoes and diets high-in-lycopene is weaker than in American or European populations. Given that the intake of carotenoids in this study was much lower than in American studies, it may even suggest some U-shaped curve of dependency. The narrow range of the study allows us only to state that LYC might protect from low-grade PC in the Vietnamese population.The last study of LYC was the North Carolina–Louisiana Prostate Cancer Project (PCaP), a cross-sectional study investigating, separately, the role of LYC in European Americans (EA) and African Americans (AA), aged 40–79 years. Dietary habits were collected based on the National Cancer Institute’s Diet History Food-Frequency questionnaire (NCI-DHQ), containing 144 common food items. Additionally, data about carotenoid supplement intake were taken. Results were presented as tertiles . In this study, there was a protective effect of LYC against aggressive PC (defined as Gleason ≥ 8) and the OR for the third tertile was 0.55 . However, it referred only to the EA group; as for the AA, they found no significant associations . Due to Increased consumption of LYC from canned or cooked tomatoes might be related to a reduced hazard of developing PC  and, supposing, aggressive PC;Intake of more than 10 mg of LYC was related to a reduced risk of diagnosing PC, by at least 10%, compared to an intake below 3.6 mg per day in European and American populations;For men with TMPRSS2: ERG translocation, the reduction in risk of diagnosing PC reached about 50%;LYC was associated with an increased risk of developing PC without abnormal PSA or DRE by about 40%—however, this subgroup of PC is actually undetectable (by definition) due to the lack of clinical signs;The clinical significance of underdiagnosed PC cases must be evaluated—if they remain unharmful over time, it would be beneficial from a public health point of view not to detect their presence—reducing the postoperational burden of patients and costs of healthcare;In the Vietnamese population, an intake of 1.2 mg of LYC per day was associated with a reduced risk of diagnosing PC by about 50% compared to a daily intake of less than 0.648 mg.When summarizing our conclusion for LYC it can be stipulated that:There have been twelve studies that focused on BC. However, their comparability is low. Two were cross-sectional ,28, fivep < 0.001). Among these cases, significantly higher concentrations of α-linoleic (ALA) were observed, which was meant to be a covariate for results (elevated in PC cases). The difference in plasma BC levels was also significant, although less remarkably. Mean concentrations were 24.7 and 20.4 µg/dL, respectively (p < 0.04), however BC concentration varied, depending on the month of sampling, with highest concentrations between July and September. This study observed a great increase in PC risk for higher BC concentrations. The RR for the third tertile in comparison to the first was 2.29  in a fully adjusted model , BMI, years of smoking, alcohol consumption, education, physical activity, serum total cholesterol, and serum ALA) [Another large-scale study also investigating the Finnish population was The Kuopio Ischaemic Heart Disease Risk Factor Study (KIHD). This trial evaluated serum levels of BC in patients aged 42–60 years at baseline. The second measure was taken at the end of the study. Mean concentrations were divided into tertiles . This was the only study that provided full data on the blood sampling procedure, which allowed us to largely exclude diagnostic bias in this study. A total of 68 PC cases were registered. In this study, cases were significantly older than those without PC—means for age were 58.9 and 55.9, respectively (rum ALA) . This nep-value—suggesting again an inverse association between BC and cigarettes. Interaction with SNPs turned out to be beneficial for the TC/CC genotype of rs699473 in the SOD3 gene. The OR value for the risk of high grade PC in Q4 versus Q1 among TC/CC genotype carriers was 0.20 (95% CI: 0.09–0.45) [The fourth study was conducted by Nordström et al. and analyzed circulating carotenoids, PC risk, and their relation to the genomic instability in a population of 81 Caucasian men. The concentrations were given in quartiles. In total, 20 of the SNPs in SOD1-3, XRCC1 and OGG1 genes were measured. In this study, men with high-grade tumor (Gleason score ≥ 7) were preferably chosen. Comparing the highest versus the lowest quartile, OR for diagnosis of a high grade of PC was 0.31  in the fully adjusted model (adjustment for age at diagnosis (years) and circulating cholesterol (mg/dL), smoking status at diagnosis (ever vs. never), and Caucasian origin). It is worth noting that nonadjustment for smoking resulted in both higher OR value and a lower In summary, none of the prospective studies met the requirements to be used as a source of any strict conclusions . Simultaneously, the study by Nordström et al. comprised not enough cases and its retrospective character could be a source of bias. Based on the collected data, we can only derive certain partial conclusions:1. In smokers, constant supplementing 30 mg of BC  each day may decrease the risk of developing nonaggressive PC;2. Maintaining a higher serum BC level might decrease the risk of high grade PC, particularly among people with the TC/CC variant (rs699473) in the SOD3 gene. On one hand, SOD3 plays a significant role in ROS handling. On the other hand, free radicals react easily with BC. It could provide an explanation for the correlation of SOD3 polymorphisms to protective effects of BC.Nine studies included in our review evaluated the effectiveness of retinol toward the prevention of PC. These included one cross-sectional , five cap < 0.0001). In fact, exclusion of participants diagnosed within two years of blood sample collection  slightly increased HR [Three case-control studies referring to serum concentration of retinol were nested in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) within the ATBC study and in the PCPT. The PLCO involved 692 cases of PC . Concentrations were given in quintiles . Statistically significant results were found for risk of high-grade disease (Gleason score ≥ 7) in Q1 versus Q5, with an OR of 0.52 . For each quintile, a protective effect was shown (compared to the first one). A correlation with clinically aggressive disease stage (III or IV) could not be demonstrated. What weakens the impact of the results from this study is that retinol concentration was measured only once, at the baseline of the study. For a small subgroup of 46 men, assessment was repeated one year after baseline and only a moderate correlation with initial retinol concentration was found (r = 0.38) . The ATBeased HR .The PCPT design was also described in the previous chapter. In this investigation by Nash et al., a positive association of serum retinol and aggressive PC was shown. At the end of the study, all participants underwent prostate biopsy, resulting in 54.1% of total PC cases. Measurements of retinol concentrations were done annually. Average results were given as quartiles . An increased risk of total PCa, as well as aggressive disease (Gleason score ≥ 7), was shown—ORs (Q1 versus Q4) were 1.43  and 1.91  respectively. Moreover, results were stratified depending on whether PC was detected for cause (indicated by DRE or PSA) or not for cause. The highest serum retinol levels resulted in a 145% increased incidence of aggressive PCa . All these associations were seen only in the placebo arm of the PCPT (men who had not been receiving finasteride) . As it wIt is of high importance to mention the discoveries of novel research conducted with participants of the PLCO study . From 31 investigated genes involved in retinol metabolism , whether it merely causes increases in PSA concentrations, or enables a more sufficient primary prevention .The relationship between carotenoids and PC appears heterogeneous and complex. Results of the largest, prospective trials suggest that dietary LYC is protective against PC. For other carotenoids, such as BC and retinol, the relation becomes less obvious, though there is accumulating evidence suggesting that retinol, at least in subjects with distinct ALDH1A1/3 SNPs, exerts adverse effect.Molecular and genetic differences may result in varying response to carotenoids. Lifestyle and ethnicity seem to strongly modify it as well. Finally, the significance and diagnosis of PC depends strongly on primary prevention methods. Therefore, the vast majority of discussed studies were, in fact, more directly related to PSA than to PC itself. Discussions for AC, zeaxanthin/lutein, and β-cryptoxanthin will not be carried out in the present review as there are insufficient data on their effectiveness in reducing the risk of PC to perform any comprehensive analysis.Apart from their influence on cancer risk prevention, carotenoids are vastly researched in the area of cancer treatment, after the development of disease. The aim of this section was to discuss the effects of carotenoid intake on PC treatment outcomes. In our search for trials referring to this question, we identified 19 papers that fitted the inclusion criteria as described in the method section. Among those, there were four clinically controlled trials, three cohort studies, one cross-sectional study, and five case-series reports. Two studies were nested in large randomized controlled studies (RCTs) and one was nested in a cohort study. Apart from this, we identified six experimental animal trials that evaluated the influence of carotenoids on PC tumor growth. Due to the lack of consistent methodology and endpoints, we did not perform a meta-analysis of the gathered papers .There have been no consistent results favoring positive effects of carotenoids in PC treatment. Moreover, it must be considered that the existence of statistically significant outcomes in studies may not reflect the real association, but also bias, resulting from performing additional analysis in the pursuit of positive results.Furthermore, only one of the clinically controlled trials, carried out by Margalit et al. , was of Regarding cohort studies, the one conducted by Watters et al.  seems toAs other papers were either case series or animal studies ., there In an RCT by Paur et al. , the effIn another interventional trial, Grainger et al. found no significant difference in PSA slopes between PC patients receiving tomato-soy juice prior to the prostatectomy and a control group . Wang etcis-retinoid acid (13cRA). However, as their work did not focus on retinoids themselves, no strong conclusion may be drawn from their study for this review. Similarly, Di Bella et al. [cis-retinoic acid.Apart from assessing their role as intervention agents in cancer treatment alone, some attention has been drawn to the potential of employing carotenoids in combination therapy. DiPaola et al.  conductea et al.  utilizeda et al.  reportedtrans LYC to the regimen. The unambiguity seen between trials in animals and humans may originate from uncontrolled environmental influence in the latter case. This is consistent with an analysis performed by Druesne-Pecollo et al. [More conclusive results may be drawn from experimental animal testing. Among six of the reviewed studies, two carried out by Lindshield et al.  and Rowlo et al. , who furo et al. . On the o et al. . Those to et al. .In conclusion, current evidence does not allow drawing conclusions regarding the relation of carotenoids and PC prognosis. However, the aforementioned research highlights the need for careful evaluation of differences in action of distinct carotenoids in distinct populations and even among different patients. Furthermore, unambiguity between studies exist, especially between humans and animal models, emphasizing the importance of confounding factors.-trans geometric configuration, tissue LYC is present primarily as cis-isomers [All the human studies identified in regard of this topic referred to the LYC. Commonly consumed tomato products, such as tomato soup, sauce, and juice in daily servings, can increase and prostate LYC concentrations in men with PC after three weeks of a diet rich in tomato products . While t-isomers .Prostatic hyperplasia (PH) develops in the majority of men along with the ageing process. Approximately 80% of men have PH at the age of 80. PH can result in urethral obstruction and lower urinary tract symptoms. The primary cause is the increased sensitivity of the prostate to dihydrotestosterone (DHT) . MoreoveSalmonella enteritidis lipopolysaccharide (LPS) was compared to the effect observed in vivo in the prostate of rats with partial bladder outlet obstruction. As expected, LPS induced a proinflammatory phenotype in macrophages. LYC, Se, and SeR inhibited the inflammatory cascade, but it was the LYC-Se-SeR combination that caused a greater inhibitory effect on the expression of COX-2, 5-LOX, and iNOS. The LYC-Se-SeR association was effective in reducing the loss of IκB-α, NF-κB-binding activity, mRNA levels of TNF-α, malondialdehyde, and NO content [®) reduced the extension and grading of inflammation, with a significant decrease in inflammatory cells markers such as CD3, CD8, CD20, and CD68 [Clinical trials are in compliance with the results from experimental studies discussed in part I of this review . In a ra content . In a muand CD68 .Cucurbita pepo, Epilobium parviflorum, Pygeum africanum, and Serenoa repens underwent a short-term phase II randomized double-blind placebo controlled clinical trial. Tested as a three-month intervention for the management of symptoms of medically diagnosed BPH in 57 otherwise healthy men aged 40–80 years, it reduced both daytime and night-time bladder emptying frequency. In the active group, a significant reduction in the median of the validated international prostate symptom score (IPSS) was observed [It is worth mentioning that there have been some clinical trials evaluating treatment efficacy of other medical herbal preparations containing LYC. For instance, ProstateEZE Max, an orally dosed herbal preparation containing LYC, observed .LYC has some of the strongest antioxidant properties (in terms of quenching singlet oxygen) of carotenoids. Compared to BC, LYC has been reported to be twice as effective and 10-fold more active than α-tocopherol . The effIn an attempt to check the impact of LYC on soluble receptors for advanced glycation end products (sRAGE) in seminal and blood plasma, a randomized controlled trial was carried out. The study included 15 fertile volunteers and 13 normospermic male partners from infertile relationships. The 12-week administration of 20 mg of LYC or placebo was followed by a crossover and treatment for a further 12 weeks. sRAGE levels were determined using ELISA. LYC decreased sRAGE in seminal plasma, but not in blood plasma. A selective local uptake of LYC in the male reproductive tract, namely in the prostate, was suggested. Suppression of oxidative stress by LYC may explain, in part, the putative improvement in fertility reported after LYC treatment, as reactive oxygen species are known to oxidize sperm membrane lipids, proteins, and nucleic acids, leading to cellular dysfunction and subsequent male infertility .This review presented insight into the recent findings about the influence of carotenoids and retinoids on prostate physiology and pathology, with special concern given to PC and prostatic hyperplasia. A strong potential of LYC in the prevention of PC is suggested, which seems to be increased in certain populations of men with TMPRSS2: ERG. For other carotenoids, sufficient evidence is lacking, and only under certain situations we may suspect an impact on PC . Furthermore, regarding the treatment of PC, their roles remains still uncertain.Serenoa repens and Se, counteracts prostate hyperplasia due to its proapoptotic, anti-inflammatory, antiandrogen, and antioxidant activity. Other properties of carotenoids, although being less frequently examined, are very promising, with a good example of astaxanthin and its apparently selective activity against PC cells, combined with a protective effect on normal prostate epithelial cells. However, as most studies were carried out with cell models, employing very high and nonphysiologic concentrations, it remains to be elucidated whether similar effects may occur in vivo.LYC, applied alone, or in combination with The diversity of carotenoids and their influence on the human organism and prostate in particular still remains a source of fascinating, surprising findings. Undoubtedly, numerous discoveries in this field are awaiting us in the following years.


Apolipoproteins, the key components of lipoproteins, play vital roles in the combination and transportation of lipids. Numerous research articles have accumulated solid evidence that lipoproteins are closely related to various types of tumorigenesis. In this review, we focused on the associations between several apolipoproteins and breast carcinoma and distinguished the effects and significance of apolipoproteins in different locations to validate their roles in breast carcinoma development. For example, apoD and apoE in serum are viewed as risk factors for breast carcinoma. ApoD, apoE and apoA-I in mammary tissues inhibit tumor growth. Moreover, apoB, apoJ and apoA-I have the potential to function as diagnostic or prognostic markers in the clinic. ApoEdp and apoJ treatment on breast carcinoma could significantly restrict tumor growth. In general, the aim of this review was to further analyze the associations between some members of the apolipoprotein family and breast cancer. Apolipoproteins are named for their association with lipids and their roles in lipid transport. Plasma apolipoproteins bind to the surface of lipoproteins and fulfill multiple functions in lipid metabolism , such asIn recent decades, together with the improved standards of living and increased life expectancies, epidemic studies have reported associations among obesity, lipoproteins and malignancy. One report conducted by the International Agency for Research on Cancer (IARC) Working Group on Body Fatness in 2016 suggested that body fat is sufficiently linked to risks of more than a dozen cancers , 9. ObesIn 2018, there were more than 2 million new BC cases and ~ 0.63 million BC-related deaths worldwide. In Asia, BC incidence reaches 0.9 million, accounting for 43.6% of the total new cases worldwide, and the mortality is approximately 0.31 million, accounting for 49.6% of the total global BC-related deaths . A hiera2 concentrations distinctly reduce apoD gene expression and excretion, which inversely induces the proliferation of ZR-75-1 BC cells at the molecular level [ApoD, also known as gross cystic disease fluid protein-24 (GCDFP-24) and progesterone-binding cyst protein (PBCP), is a member of the lipocalin superfamily. In 1963, human apoD was detected in plasma. Ten years later, McConathy et al. originally differentiated apoD from plasma HDL . The apoar level . In addiar level . All of ar level . As earlar level . Later, ar level . In 2001ar level . Among Bar level , and it ar level . Howeverar level , 35. Redar level , which cER pathways: Estrogen and its receptor promote gene expression and BC cell progression in the nucleus through the genomic pathway. The MISS-ER pathway promotes the cell cycle by interacting with the MAPK pathway via Ras. MAPK pathway: MAPK translocation from the cytosol into the nucleus enhances cell cycle progression and cell proliferation. Abundant apoD in the cytoplasm binds with MAPK and blocks its translocation, which consequently has an inhibitory effect on BC cell proliferation. ApoD is the second factor that combines ER signaling and the MAPK pathway due to the reduced apoD expression induced by ER. PR pathway: PR and the PR receptor (PRR) exert the same influence as ER on gene expression and cell proliferation. When apoD binds to progesterone with a strong affinity, some PR is allocated to the inactivation of metabolism. On the other hand, apoD may produce a “slow release” effect on PR and suppress gene expression in the nucleus, which also hinders the proliferation of BC cells. COX-2 and 5-LO pathways: Free AA promotes the COX-2 and 5-LO pathways. Prostaglandins and leukotrienes are the end products of the two pathways, and they inhibit apoptosis, facilitate neo-angiogenesis and stimulate cell proliferation. Prostaglandins not only promote angiogenesis but also induce aromatase, which converts androgens into estrogens. When apoD closely chelates with free AA, the downstream products are reduced, and the malignant reactions are subsequently avoided. ER decreases apoD expression, and the final effects of apoD on BC cells may depend on the expression or binding capability with the above cytokines.There is an interesting saying that “apoD is a good citizen but a bad neighbor”, which may be attributed to apoD symbolizing cell senescence. When apoD is present in cancer cells, it promotes the senescence of tumor cells and inhibits cell proliferation. Upon apoD becoming abundantly present in stromal cells, the senescence of these stromal cells is not a good sign due to the decrease in something that could otherwise restrain the invasion of tumor cells, such as tissue inhibitors of metalloproteinases and serine . In addiApoE is located on chromosome 19q13.2 and consists of four exons that lie on the long arm of chromosome 19. On the chromosome, the apoE gene encodes three common alleles, including ε2, ɛ3 and ɛ4. The liver synthesizes approximately three-quarters of plasma apoE. ApoE is also secreted by numerous other tissues, such as the kidneys, ovaries, testes, skin and macrophages . A liverApolipoprotein A-I, a subtype of apoA, is a cofactor of LACT and plays a role in reverse transporting cholesterol from peripheral tissues to the liver. The liver and small intestine synthesize this subtype of apolipoprotein . CompareApoJ, known as clusterin, is involved in lipid transport, cell adhesion, programmed cell death and complement cascade reaction. The major actions induced by apoJ prevent cell death. In carcinoma development, apoJ inhibits the cancer cell apoptosis and then has effects on tumor growth and metastasis . RecentlAs previously mentioned, the apolipoprotein family contains numerous other members, such as apoB, apoC and apoM. These apolipoproteins are not as closely associated with BC as apoD and apoE. Therefore, we will review the existing or potentially existing hallmarks of these apolipoproteins in the carcinogenesis and development of BC. First, the associations among apoB, apoC-I and BC will be described, which are supported by existing literature. Second, we will aim to provide a theoretical conjecture regarding the potential roles of apoM in BC.ApoB transports lipids into cells in the human body , and theApoM, isolated from chylomicrons, was first identified by Xu and Dahlback at the end of the twentieth century. ApoM is primarily bound to HDL particles and is present on low levels in LDL particles . ApoM haApoM carries S1P to bind with S1PR, which maintains the stability of the S1P concentration in plasma. S1P inhibits the invasion of breast tumor cells. Increased Vitamin D (Vit D) expression induced by apoM and its receptor (VDR) modulates the autophagic process and cell death in BC cells and normal mammary tissue. Furthermore, apoM promotes the maturation of HDL from pre-β-HDL. A high concentration of HDL-C lowers the risk of BC. However, the diverse existence of SR-BI, a receptor of HDL, in BC tissues may change BC development by affecting tumor cell anti-apoptosis and angiogenesis via the Akt and ERK1/2 pathways. Consequently, the roles of apoM in BC are worthy of exploration.Apolipoproteins bind to the surface of lipoproteins and have a role in transporting and metabolizing lipids. Various apolipoproteins form the diverse lipoproteins. These apolipoproteins are not only involved in coronary-artery disease, atherosclerosis, MS and T2D but also participate in various types of carcinoma. The present review discussed the roles of apolipoproteins in BC and summarized the different effects of apolipoproteins in this disease Table . In term


Chronic rhinosinusitis (CRS) is an inflammatory disease of the paranasal sinuses. It is defined as the presence of a minimum of two out of four main symptoms such as hyposmia, facial pain, nasal blockage, and discharge, which last for 8–12 weeks. CRS significantly impairs a patient’s quality of life. It needs special treatment mainly focusing on preventing local infection/inflammation with corticosteroid sprays or improving sinus drainage using nasal saline irrigation. When other treatments fail, endoscopic sinus surgery is considered an effective option. According to the state-of-the-art knowledge of CRS, there is more evidence suggesting that it is more of an inflammatory disease than an infectious one. This condition is also treated as a multifactorial inflammatory disorder as it may be triggered by various factors, such as bacterial or fungal infections, airborne irritants, defects in innate immunity, or the presence of concomitant diseases. Due to the incomplete understanding of the pathological processes of CRS, there is a continuous search for new indicators that are directly related to the pathogenesis of this disease—e.g., in the field of systems biology. The studies adopting systems biology search for possible factors responsible for the disease at genetic, transcriptomic, proteomic, and metabolomic levels. The analyses of the changes in the genome, transcriptome, proteome, and metabolome may reveal the dysfunctional pathways of inflammatory regulation and provide a clear insight into the pathogenesis of this disease. Therefore, in the present paper, we have summarized the state-of-the-art knowledge of the application of systems biology in the pathology and development of CRS. Chronic rhinosinusitis (CRS) is an inflammatory disease that involves the accumulation of mucosa in the nose and sinuses. It can often lead to allergic rhinitis (AR), which significantly reduces a patient’s quality of life ,2,3,4. TSystems biology uses a holistic approach to study the complex interactions in biological systems based on the analysis of genome, transcriptome, proteome, and metabolome . In addiCFTR) gene, impairing respiratory mucosal function [The development of CRS can be influenced by numerous factors, of which genetic predisposition is the leading one. According to the current literature, some evidence emphasizes the association between CRS and mutations in the cystic fibrosis transmembrane regulator . Genetic strategies are usually applied as hypothesis-driven approaches or in GWAS. The first approach relies on a known a priori candidate gene apparently associated with the diseased state or theoretically involved in its pathogenesis. This approach compares the allele frequencies of SNPs in the studied group vs. controls [In addition, the CRS genetic background can be linked with the genes associated with bacterial colonization, immunological processes in the body, primary ciliary dyskinesia (PCD), or taste receptor (T2R) genes  in whom common CF mutations were found were excluded from further study. In addition, the authors detected CFTR variants  of the splice acceptor site in intron 8 and F508C, 1507V, and 1506V polymorphisms of the CFTR gene. Moreover, the CFTR gene exons and flanking introns were analyzed by denaturing gradient gel electrophoresis. The obtained data were statistically analyzed using univariate statistics (Student’s t-test and Chi-square test). Consequently, the authors found that 9 out of the 10 CF carriers had the M470V polymorphism. Moreover, M470V homozygotes were over-represented in the 136 CRS patients vs. controls (p = 0.03). Therefore, the authors concluded that mutations in the CFTR gene could predispose to CRS. Furthermore, the association between the CFTR gene and CRS was proposed by Pinto et al. [7q31.1–7q32.1 related with the CFTR gene can be associated with the development of CRS. Other studies also confirmed the observed increase in the frequency of CFTR mutations in CRS patients, suggesting that populations with CRS are at a higher risk of being the carriers of a CFTR mutation [CFTR-mediated chloride transport to improve mucociliary clearance in CRS patients [CFTR mutation. With the use of this agent, the authors observed the medical reversal of chronic sinusitis in CF patients [Wang et al.  assessedmutation ,29. Therpatients . AnotherIn terms of hypothesis-driven theory, there is also evidence that PCD, the autosomal recessively inherited disease affecting the structure and function of cilia, can lead to CRS . PCD alsTAS2R38 gene at positions 49, 262, and 296, which can encode functional amino acids  and nonfunctional ones . Patients with PAV/PAV, PAV/AVI, and AVI/AVI genotypes were characterized as supertasters, intermediate, and nontasters, respectively. The supertasters group can effectively produce nitric oxide, have faster mucociliary clearance, and exhibit a more potent bactericidal effect compared to persons with PAV/AVI and AVI/AVI genotypes [TAS2R38 gene may be a future therapeutic target to promote T2R38 innate immune response in each subgroup of CRS patients.Some investigations concern the association of bitter taste receptors (T2Rs) and CRS. Bitter taste receptors are located in the human tongue and in the bronchial and sinonasal epithelium. When stimulated with bitter agonists, these receptors increase the intracellular calcium level, leading to increased production of nitric oxide. Such a cascade of reactions increases mucociliary clearance and induces an antibacterial effect, ultimately bringing significant relief to CRS patients . In theienotypes . AdditioHLA) should be underlined. HLA alleles such as HLA-A_24, HLA-A_74, HLA-B_54, HLA-B1_3, HLA-B1_08, HLA-B_07, HLA-B_57, HLA-Cw_04, HLA-Cw_12, HLA-DRB1_03, HLA_DRB1_04, and HLA-DQB1_03 have been described in the literature as associated with CRS, mainly with its polyposis subtype [NOS2A). The authors found that polymorphisms in the NOS2A gene were statistically significantly associated with the eosinophilic form of CRS with nasal polyps (CRSwNP) [Regarding immunological genes, numerous studies have been performed focusing on the association between the studied immunological gene and CRS, among which the human leukocyte antigen  .As was mentioned earlier, instead of searching for SNPs in a known a priori gene, which is suspected to be related to the disease state, GWASs and their alternatives, pGWASs, have been successfully performed.p73 gene. In total, 206 CRS patients and 196 controls enrolled in the study. The DNA from CRS patients was extracted from the peripheral blood leukocytes, while in the case of controls, DNA was extracted from the blood (n = 30) or saliva samples (n = 166). The samples were pooled separately for CRS and control groups and subsequently genotyped by high-density SNP genotyping study. The pooled DNA was hybridized and interrogated with 555,175 SNPs. Subsequently, a subset of highly ranked SNPs most likely associated with CRS was selected for individual genotyping, which was carried out on 1536 SNPs, among which two SNPs from the p73 gene were evaluated (rs3765731 and rs3765692). In addition, the authors sequenced the entire p73 gene in 10 patients and 1 control. This sequencing did not identify any mutations capable of altering amino acid content in the gene product. Statistical analyses showed that one SNP from gene p73 (rs3765731) was significantly associated with CRS (p < 0.05). The authors noted a significant difference in the minor allele frequency between the studied groups. The minor allele A was more frequently identified in the control group, while the minor allele G was more associated with CRS. Additionally, it was found that the homozygous AA patients had a seven-fold lower risk of CRS compared to GG homozygotes, and the minor allele A apparently had a protective effect in comparison with allele G. Further studies are also required to evaluate the involvement of p73 gene in some intracellular signaling to effectively explain CRS development.In the work of Tournas et al. , a pGWASStaphylococcus aureus colonization by high-density genotyping that interrogated one million SNPs. The 39 SNPs that were most likely associated with bacterial colonization were selected for further individual genotyping. Among them, 23 SNPs were significantly associated with S. aureus colonization (p < 0.05). In total, 12 SNPs were associated with an increased risk for S. aureus colonization, and 11 SNPs were correlated with a protective effect against this bacterium. The authors also used the Ingenuity Pathway Analysis (IPA) software to evaluate if genes mapping close to the SNPs mentioned earlier belonged to the same functional pathway. The analysis showed that the top biological functions were related to cell-to-cell signaling, cell morphology, growth, and proliferation.A pGWAS was also performed by Cormier et al. . The autRYBP, RAC1, FAM79B, and CACNA2D1. These genes are known to be involved in endocytic internalization and bacterial recognition. Therefore, the authors suggested that further studies on these genes and their biological functions are required as they would be promising in the search for novel strategies for CRS therapy.Moreover, it was found that the top canonical pathways were related to IL-8 and IL-3 signaling, micropinocytosis signaling, and the mammalian target of rapamycin signaling. The authors observed that selected SNPs were mapped within or close to 21 genes including Summing up the current knowledge on the genetic susceptibility to CRS, we can assume that either identification of SNPs among the known genes or search for new SNPs using a GWAS strategy would lead in the future to new target avenues of CRS and aid in better understanding the disease pathogenesis. Analyses of protein components in biological fluids, such as the blood (serum and plasma), urine, saliva, cerebrospinal fluid, and nasal mucus, can increase the current knowledge on the biology and physiology of the conditions of human body ,49,50,51In addition, the improvements in proteomic technology could lead to the search for safer, more effective, and new drugs. Casado et al.  comparedA comprehensive examination of NLF from 10 volunteers, collected before and after they had been submitted to nasal provocations, was performed by Casado et al.  using thFrom inhaled air, the airways are confronted with a variety of pathogens required to be filtered and transported away. Nasal mucus is the first-line defense barrier and functions as a physical barrier against particles, irritants, microbes, viruses, food, and liquids. Hairdressers have an increased risk of developing respiratory symptoms from the airways such as asthma and rhinitis, as they are exposed to many reactive chemicals that can induce immunological reactions. Mörtstedt et al.  analyzedp < 0.05) abundant and 104 were significantly lower in the CRS cohort compared to healthy controls. For a better understanding of the molecular mechanisms underlying respiratory diseases, Biswas et al.  correlatThey identified 7 of the 10 targeted proteins in the MRM mode, many of which were related to innate and acquired immunity. Quantitative analysis showed differential expression of some proteins in CRS patients compared to the control subjects, which included lysozyme C, IGLV4-3 (immunoglobulin lambda variable 4-3), bactericidal protein, calgranulin, lipocalin, mucin B, and deleted brain tumor protein . The patInterestingly, DMBT1 protein is closely associated with MUC5B in the respiratory tract secretions. Based on the reviewed literature, many proteins that are significantly elevated in the CRS sinonasal secretions are implicated in the airway inflammatory reaction and immune response . Some ofMetabolomics aims at qualitative and quantitative analysis of low-molecular-weight endogenous compounds (up to 1500 Da) in biological samples . The observed levels of metabolites result from the dynamic changes in the genome, transcriptome, and proteome. In addition, they reflect the current phenotype of a particular organism. Therefore, it is considered that the metabolome is related to the genotype-to-phenotype gap ,67. ThreThe data set obtained from a targeted study is a matrix composed of only selected metabolites vs. analyzed samples, which is a much simpler one compared to the matrix acquired in an untargeted approach. Therefore, the pretreatment and analysis of data are less laborious and a similar type of statistical method is used for both approaches. As a result of the targeted metabolomic study, practically only a few out of previously chosen metabolites appear relevant and statistically significant among the studied groups . TherefoPseudomonas aeruginosa, the pathogen responsible for various inflammation processes [Fazlollahi et al.  demonstrrocesses . Furthern = 6) with eosinophilic rhinosinusitis  and healthy controls . Using the metabolomic approach, the authors performed a targeted lipidomic study with LC-MS/MS. Briefly, the isolated eosinophils were suspended in Hank’s buffer salt solution, incubated, and centrifuged. The cell supernatant was diluted in methanol  and extracted using anion-exchange SPE columns after the addition of deuterated internal standards. The targeted analyses of various eicosanoids and docosanoids were carried out using an Acquity UPLC™ BEH C18 column  on the triple quadrupole linear ion trap mass spectrometer in negative ionization and MRM modes. In the analysis, the authors observed significantly lower amounts of both cyclooxygenase (COX) and lipoxygenase (LOX) products in the NP-EOS group compared to the PB-EOS group. Regarding COX products, lower amounts of prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), thromboxane B2 (TXB2), 12-hydroxyhepentadecatrienoic acid (12-HHT), and 11-hydroxyeicosatetraenoic acid (HETE) were observed. In the case of LOX products, lower levels were observed for 12-hydroxyeicosatetraenoic acid (12-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE), lipoxin A4, 8,15-diHETE, and 17-hydroxydocosahexaenoic acid (HDoHE). The authors also noticed a selective upregulation of LTD4 biosynthesis in NP-EOS patients. Furthermore, despite the observed downregulation of COX and LOX pathway products in NP-EOS patients, no significant changes were noted in the level of arachidonic acid, a precursor of COX and LOX, which suggests that patients with NP-EOS may display unique lipid mediator profiles that can also affect specific EOS phenotypes.In the study conducted by Miyata et al. , a multin = 54) and CRSsNP (n = 46) patients, healthy controls (n = 40), patients with AR (n = 51), and patients with nonallergic rhinopathy (n = 43). In a targeted metabolomic study, the authors used the immunoassay method for the determination of neopterin. Human monocytes and macrophages mainly produce this metabolite, and its amount corresponds with the capacity of reactive oxygen species production by these cells [t-test.The targeted analysis was also proposed by Tsybikov et al.  to assesse cells . Therefose cells . The obtp < 0.001) in CRSsNP patients compared to healthy controls in both sera and nasal secretion samples. Furthermore, neopterin levels measured in nasal secretions were also significantly elevated (p < 0.001) in CRSsNP patients compared to those with CRSwNP. Such a correlation was not observed in the case of sera samples. In addition, no significant correlation was found between the neopterin levels among the other CRS groups. Therefore, this metabolite would be proposed as a potential biomarker only for CRSsNP patients. To confirm those findings, the analyses should be subsequently carried out on a larger group of patients using more sensitive analytical instrumentation involving, e.g., MS detection.The levels of neopterin were significantly elevated  and a test set (30%) for method validation. The authors compared four binary relations and assessed both false-positive and false-negative and true-positive and true-negative results. The correlated data were as follows: CRS vs. NP, CRS vs. controls, NP vs. controls, CRS, and NP vs. controls. The accuracy of the training set was in the range of 82–84%, whereas for the validation (test) set, it ranged from 67% to 90% depending on the data compared. A sensitivity of 100% was achieved for all sick patients (NP and CRS vs. control), whereas a 100% specificity was achieved for the CRS vs. NP group. Thus, the preliminary results seemed to be promising; however, as the authors noticed, the proposed approach requires that more patients should be enrolled in the study. Furthermore, the authors did not identify the VOCs responsible for the obtained recorded change in electrical resistance, so untargeted metabolomics followed by identification should be carried out for VOCs.A different approach was proposed by Broza et al. . The autn = 54) and CRSsNP (n = 34) as well as from healthy controls (n = 20). Patients with CRS were also classified based on atopic presence. The classification of patients in the CRS subgroup was performed after nasal endoscopy examination and sinus computed tomography. In contrast, atopic presence was evaluated with a skin prick test using 21 aeroallergens. The breath samples were collected using a handheld portable NIOX device . The obtained data sets were evaluated using univariate statistics . The results showed that the mean nNO level in healthy controls was significantly higher than that in both groups of patients (CRSwNP and CRSsNP) (p < 0.001). In addition, patients with CRSsNP had a significantly higher level of nNO compared to patients with CRSwNP (p < 0.001). Regarding atopic subgroups, the nNO levels were markedly higher in both CRS groups of patients with atopy and nonatopic patients (p < 0.001). The area under the receiver operating characteristic curve for all studied groups  was in the range from 0.83 to 0.988 with sensitivity and specificity ranging from 0.83 to 0.95 and from 0.70 to 0.97, respectively. Therefore, the exhaled nNO level could be used to differentiate CRSwNP patients from those with CRSsNP, although the atopic status of a patient also has some influential role in changing the nNO level. To confirm such preliminary results and to evaluate the significance of the presence of atopy as well as the potential role of nNO as a diagnostic and monitoring marker, the study should be validated in a larger group of both CRS patients and healthy controls. A summary of the results from metabolomic studies is presented in Exhaled breath samples were also examined by Liu et al. . The autCFTR gene, which affects the mucociliary transport impairment. Additionally, genes encoding HLAs and involved in arachidonic acid metabolism may contribute to the pathogenesis of CRS. The genetic background of this disease can also be related to the genes responsible for bacterial colonization, immunological processes in the body, PCD, or taste receptor genes. Therefore, the future direction of genomic analyses should confirm these findings on extensive, well-designed studies with detailed patient phenotyping. Moreover, other potential concomitant diseases  should be genetically studied as pathogenic mechanisms other than CRS. Furthermore, a new therapy should be developed targeting the genes of interest, such as ivacaftor, which as mentioned in this review target the G551D-CFTR mutation and improve mucociliary clearance in CRS patients. Besides the mentioned proteomic studies allowing to create protein profiles that can differentiate patients with CRS vs. controls, some of these proteins may be useful as potential CRS biomarkers, e.g., mucin glycoprotein, for further research. Nasal and sinus secretions in children with CRS may contain an excess of MUC5B compared to other mucin glycoproteins. The nasal mucus, as shown by various proteomic studies, could be a source of biomarkers. Among many different proteins identified as important in CRS based on the current literature (Chronic sinusitis significantly reduces a patient’s quality of life. Treatment for this condition is mainly focused on combating local infection/inflammation or improving sinus drainage. There is still a need to search for new indicators directly responsible for the pathogenesis of the disease. The application of systems biology seems to be promising mainly because it concentrates on genetic, proteomic, and metabolomic levels. Due to the use of advanced analytical instrumentations, libraries, and bioinformatics methods and the examination of the vast amounts of data obtained from the analysis of the genome, transcriptome, proteome, and metabolome, the omics can be interpreted and assessed. The current genomic studies confirm the relationship observed between CRS and mutations in the terature , some sh


Nickel is a transition element extensively distributed in the environment, air, water, and soil. It may derive from natural sources and anthropogenic activity. Although nickel is ubiquitous in the environment, its functional role as a trace element for animals and human beings has not been yet recognized. Environmental pollution from nickel may be due to industry, the use of liquid and solid fuels, as well as municipal and industrial waste. Nickel contact can cause a variety of side effects on human health, such as allergy, cardiovascular and kidney diseases, lung fibrosis, lung and nasal cancer. Although the molecular mechanisms of nickel-induced toxicity are not yet clear, mitochondrial dysfunctions and oxidative stress are thought to have a primary and crucial role in the toxicity of this metal. Recently, researchers, trying to characterize the capability of nickel to induce cancer, have found out that epigenetic alterations induced by nickel exposure can perturb the genome. The purpose of this review is to describe the chemical features of nickel in human beings and the mechanisms of its toxicity. Furthermore, the attention is focused on strategies to remove nickel from the environment, such as phytoremediation and phytomining. There a the ash .Pycnandra acuminata, first known as Sebertia acuminata) has extraordinary ability to accumulate Ni in its latek of blue-green color, due to the presence of nickel (about 25% of the latek dry weight). New Caledonia is an archipelago with a third of its surface covered by Ni-rich soil of ultramafic rocks. Jaffrè and co-authors consider that the rich New Caledonian flora contains 2145 species adapted to ultramafic soils among which 65 are Ni hyperaccumulators [In 1976, Jaffrè et al. reported that in New Caledonia an endemic tree  appears to be one of the most abundant small organic molecules present in the latex; others Ni complexes were detected, as well as malic, itaconic, galacturonic, tartaric and aconitic acids [Analyses of New Caledonia hyperaccumulators revealed that Ni was associated with citrate complex in the cuminata ,136. Calic acids .Hordeum vulgaris), bean (Phaseolus vulgaris), cabbage (Brassica juncea), ricinus (Ricinus communis), sorghum (Sorghum vulgare), spinach (Spinacea oleracea) and tomato (Solanum lycopersicum). At the end of experiments, leaves, roots, stems, fruits or seeds were separately collected, oven-dried at 100 °C, weighed, milled and analyzed. It was shown that spinach, ricinus and cabbage were able to store nickel in the leaves. The bean, barley and tomato showed a high concentration of nickel in leaves and in stems. The bean was the most efficient in the storage of nickel in fruits or seeds and roots—unlike tomato, sorghum, ricinus and barley, which showed a storage capacity lower than that of a bean. With regard to the removal of nickel, spinach was the most efficient as it contains the highest levels of the metal per gram of dry matter with respect to the other six herbaceous crops [Alyssum murale has been developed as a commercial crop for phytoremediation/phytomining Ni from metal-enriched soils [Alyssum murale did not confer hypertolerance to cobalt. Broadhurst and Chaney [Alyssum murale (Ni hyperaccumulator), Alyssum montanum (Ni non-hyperaccumulator), and ryegrass Lolium perenne in a natural serpentine soil to affect Ni, Cu, Fe and Mn uptake. After four months with standard inorganic treatment, A. murale leaves and stems contained 3600 mg/kg Ni. Moreover, the concentration of Ni and Mn were significantly higher in A. murale respect to A. montanum or L. perenne. Both Alyssum species not accumulated Cu, while L. perenne accumulated only 10 mg/kg Cu. Besides, co-cropping A. murale with L. perenne reduced Fe and Mn concentrations in A. murale [. Psychotria douarrei and Geissois pruinosa are other plants called hypernickelophores, which accumulate more than 10,000 mg Ni/kg [Rinorea niccolifera (Violaceae) [Rinorea niccolifera collected from two sites of Luzon Island . As this species surpasses the 10,000 mg/kg Ni accumulation level in the leaves, it is regarded as a “hypernickelophore”. The studies of Roccotiello et al. highlight that Mediterranean Alyssoides utriculata leaves are Ni-hyper accumulators (higher than 1.0 g/kg) and can be used for Ni-phytoextraction purposes and for cleaning Ni-contaminated areas [Alyssoides utriculata. The results showed that the concentration of this transient metal is higher in leaves than in roots, and at the higher concentration tested (500 mg Ni/L), A. utriculata accumulates 1.1 g Ni/kg leaves as previously found. Plant water content increases significantly with Ni accumulation without affecting chlorophyll fluorescence parameters. In fact, the photosynthetic efficiency of A. utriculata is stable among Ni treatments .The experiments of Giordani et al.  studied us crops . The Ni ed soils . Nickel d Chaney  co-cropp. murale . Psychotmg Ni/kg . Fernandolaceae) ,143. Subed areas . In its ed areas  investigPhyllanthus balgooyi, Phyllanthus securinegioides (Phyllanthaceae) and Rinorea bengalensis (Violaceae) that occur in Sabah  on the Island of Borneo. This study by Van der Ent et al. showed that Ni is mainly concentrated in the phloem, in roots and stems  in all three species [Phyllanthus balgooyi the highest Ni concentration is in the phloem, but in Phyllanthus securinegioides and Rinorea bengalensis in the epidermis and in the spongy mesophyll . All three species have a highly distinctive tissue Ni distribution patterns with extreme levels of accumulation in the phloem of the root and stem. The phloem tissues in the main stem of all three species are green, due to the exceptionally high concentration of Ni2+ ions, and appear to act as a ‘sink’ with Ni concentrations reaching up to 2.1% in Rinorea bengalensis and up to 16.9 wt%, in the phloem sap from Phyllanthus balgooyi.The bioaccumulation and distribution of Ni were also elucidated in three plant species:  species . HoweverPseudomonas fluorescent [Bacillus cereus [Saccharomyces cerevisiae [Trichoderma atroviride strains F6 [Many strategies have been used for solving the problem of heavy metals pollution in the environment. The bioremediation methods for decreasing the amount of heavy metals in the environment have attracted the attention of many researchers. Plants, bacteria, fungi and algae are usually used for bioremediation of heavy metals , and in orescent , Bacillus cereus , Saccharrevisiae , and filrains F6 .Bacillus subtilis 117S and Pseudomonas cepacia 120 S to remove Ni from bacterial biomass, sludge, tea and sawdust. The authors of this study have shown that the biosorption capacity of nickel by bacterial biomass was greater than that by sludge, tea and sawdust. Moreover, the nickel removal increased with contact time from 1 to 8 h without increasing until 24 h; biosorption efficiency of nickel also increased with pH changes from 2 to 7 and remained constant thereafter. The maximum sorption efficiency of nickel was obtained at 37 °C, while at 45 °C and 55 °C the nickel biosorption was reduced.In their studies, Abdel-Monem et al.  used BacPhytoremediation has the advantage that contaminants may be treated in situ; while the major limitation is that it requires a long-term commitment, since the process is dependent on the plant ability to grow and thrive in an environment that is not always appropriate for normal plant growth.Nickel is a metal of widespread distribution in the environment. Contact with soluble and insoluble nickel compounds can cause a variety of side effects on human health. Human exposure to Ni may occur through food, water or air. Workers in Ni producing and processing industries are exposed by inhalation, and to a lesser extent, dermal contact. The nervous system is one of the main target organs for Ni toxicity; in fact, it can be accumulated in the brain. Allergy to nickel and metals is caused by the materials used in our daily life; therefore, the chances of triggering the onset of allergic reactions are high. This metal can cause an allergy that manifests as contact dermatitis, headaches, gastrointestinal and respiratory manifestations. The molecular mechanisms of Ni induced neurotoxicity are still not clear, but the researchers think that oxidative stress and mitochondrial dysfunctions have a primary and important role. Mitochondrial damage induced by Ni may occur first as mitochondrial membrane potential damage, then as mitochondrial ATP concentration reduction and finally as mitochondrial DNA destruction. Damage to mitochondrial functions interferes with the mitochondrial transport chain, amplifies ROS and exacerbates oxidative stress.2+ for Fe2+ in non-heme iron dioxygenases that are involved in DNA and histone demethylation. In vitro studies demonstrated that Ni sulphate could promote apoptosis in human epatoma cells, human T hybridoma cells human breast cancer, abrogated by curcumin. Many researchers have shown that nickel binds to amino acidic residues  of several enzymes decreasing their activity. Besides, in several enzymes the inhibitory nickel binds to an allosteric secondary site affecting activity.In the last 25–30 years, researchers, trying to characterize the carcinogenicity, due to nickel, have uncovered that epigenetic alterations induced by nickel exposure, can perturb the epigenome. DNA hypermethylation, histone modification and interference with miRNA network, and finally condensed chromatin structure create an aberrant epigenetic landscape that contributes to nickel-induced gene silencing, tumor initiation and progression. Indeed, nickel is known to cause cancer by an epigenetic mechanism, which appears to involve the substitution of NiPollution of soil, water, and air, due to heavy metals, is probably the most outstanding outcome of the evolution of our society. The reclamation of soils polluted by heavy metals can be achieved with different techniques and technologies. A possible alternative to technologies is the adoption of a biological method that consists of the use of plants that may uptake and accumulate heavy metals in their tissues. The process of metal absorption and accumulation in the plant tissues is called phytoextraction, and in the case of nickel, the phytoremediation capability, which cost is not excessive, depends on the level of nickel concentration in soil.2+ allows the Cyt C release from the mitochondria to the cytosol. Cyt C cleaves and activates caspase-9, which in turn cleaves and activates caspase-3, -6 and -7. Ni2+ promotes in the extrinsic pathway Fas and FasL interactions, leading to activation of caspase-8 and caspase-10 that activate caspase-3, -6 and -7. Caspase-3, -6 and -7 cleave PARP, which then induces apoptosis.In the intrinsic pathway, Ni