Gemcitabine (Gem) has been a standard first-line drug for pancreatic cancer (PCa) treatment; however, Gem's rapid metabolism and systemic instability  limit its clinical outcome. The objective of this study was to modify Gem into a more stable form called 4-(N)-stearoyl-gemcitabine (4NSG) and evaluate its therapeutic efficacy in patient-derived xenograft (PDX) models from PCa of Black and White patients.Methods 4NSG was synthesized and characterized using nuclear magnetic resonance (NMR), elemental analysis, and high-performance liquid chromatography (HPLC). 4NSG-loaded solid lipid nanoparticles (4NSG-SLN) were developed using the cold homogenization technique and characterized. Patient-derived pancreatic cancer cell lines labeled Black  and White  were used to assess the in vitro anticancer activity of 4NSG-SLN. Pharmacokinetics (PK) and tumor efficacy studies were conducted using PDX mouse models bearing tumors from Black and White PCa patients.Results 4NSG was significantly stable in liver microsomal solution. The effective mean particle size (hydrodynamic diameter) of 4NSG-SLN was 82 ± 6.7 nm, and the half maximal inhibitory concentration (IC50) values of 4NSG-SLN treated PPCL-192 cells (9 ± 1.1 µM); PPCL-135 (11 ± 1.3 µM); PPCL-46 (12 ± 2.1) and PPCL-68 equaled to 22 ± 2.6 were found to be significantly lower compared to Gem treated PPCL-192 (57 ± 1.5 µM); PPCL-135 (56 ± 1.5 µM); PPCL-46 (56 ± 1.8 µM) and PPCL-68 (57 ± 2.4 µM) cells. The area under the curve (AUC), half-life, and pharmacokinetic clearance parameters for 4NSG-SLN were 3–fourfold higher than that of GemHCl. For in-vivo studies, 4NSG-SLN exhibited a two-fold decrease in tumor growth compared with GemHCl in PDX mice bearing Black and White PCa tumors.Conclusion 4NSG-SLN significantly improved the Gem's pharmacokinetic profile, enhanced Gem's systemic stability increased its antitumor efficacy in PCa PDX mice bearing Black and White patient tumors.The online version contains supplementary material available at 10.1186/s12885-023-10928-w. Pancreatic cancer (PCa) is a global problem and has emerged as one of the highly fatal types of malignancies in the United States of America. It has an annual incidence of more than 62,210 cases  and a yearly mortality rate of almost 49,830 cases  and has the worst 5-year survival rate of all cancers [Also, rapid growth, early dissemination to distant organs, and resistance to chemotherapy contribute to the low survival records in PCa patients , 7. TyroCurrent treatment of PCa using chemotherapy is primarily based on nucleoside analogs. These molecules are designed to mimic natural pyrimidine, purine nucleosides, and gemcitabine (Gem), one of the analogs .2) to increase its therapeutic levels, resulting in serious side effects such as renal and hematological toxicities [As monotherapy, Gem is currently the preferred first-line therapeutic agent for advanced metastatic PCa. Modified FOLFIRINOX (mFOLFIRINOX), a combination therapy comprising of (5-fluorouracil (5FU), leucovorin, oxaliplatin, and irinotecan), albumin-bound paclitaxel  together with Gem are generally considered the optimal adjuvant chemotherapeutic regimen for PCa. Gem has improved the quality of life and increased the survival rate of patients with unresectable PCa . Howeverxicities , 16,. ThNanotechnology and its applications in drug therapy have become the fastest-growing area adopted by formulation experts to overcome challenges associated with the delivery of lipophilic or metabolic unstable drugs. Novel nano-delivery systems such as liposomes, micelles, polymeric nanoparticles, and dendrimers have recorded tremendous progress in their applications in drug delivery. Among these delivery systems, lipid-based nanocarriers are the most popular and preferred because they are highly biocompatible and biodegradable . Solid lCommercially available PCa cell lines are used frequently in animal models for drug screening and to assess the sensitivity of drugs to tumors. Nonetheless, these cells have poor predictive and translational value due to the high passages established over the past 40 years . In addiPreviously, we synthesized 4NSG from Gem and stearic acid and tested its anticancer efficacy against PCa cell lines. 4NSG remarkably reduced tumor growth in pancreatic patient-derived xenograft (PDX) mouse model with no morphological changes to mice's liver and kidney tissues .This study focuses on developing and characterizing 4NSG-SLN and testing its efficacy against patient-derived pancreatic cancer cells (PPCLs) in vitro from Black  and White (PPCL-46 and PPCL-68) patients as well as in PDX mouse models with tumors from Black and White patients.We conducted cytotoxicity and cellular uptake of 4NSG-SLN against PPCL-192, PPCL-135, PPCL-46, and PPCL-68 cells. In this study, using 4NSG-SLN, we demonstrated systemic stability of Gem, significant tumor growth inhibition in PDX mouse models in mice with Black and White PCa tumors, and the enhanced pharmacokinetic profile and improved therapeutic efficacy of Gem. This work demonstrated the first time Gem analog has been investigated in PDX mouse models bearing tumors from Black and White pancreatic cancer patients  was purchased from AK Scientific . Caprylocaproyl polyoxyl-8 glycerides (Labrasol) and Soy lecithin were obtained from Millipore Sigma Aldrich . Patient-derived primary pancreatic cancer cells from Black (PPCL-192 and PPCL-135) and White (PPCL-46 and PPCL-68) patients were obtained from Dr. Trevino's laboratory with methods previously described . All oth1  was dissolved in 100 mL dichloromethane (50.0 mL). To the solution was added tert-butyldimethylsilyl (TBS) chloride  and imidazole  successively. The reaction mixture was stirred at room temperature until Gem was consumed, as indicated by thin-layer chromatography (TLC) analysis (using 100% ethyl acetate (EtOAc)). The mixture was washed with saturated ammonium chloride, sodium bicarbonate, and sodium chloride solutions. The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under a vacuum. The TBS-protected compound 2 was crystallized from EtOAc as a white solid and used in the next step without further purification . The resulting mixture was stirred for 24 h at room temperature. After, the excess solvent was removed under reduced pressure. The residue containing intermediate 3 obtained was used in the next step without further purification.Next, to a solution of N, N-diisopropylethylamine (DIPEA) (1.200 eq) in dichloromethane (DCM) was added stearate anhydride (1.100 eq)-warmed to dissolve, and TBS-protected gemcitabine A mixture of 3  and tetra-n-butylammonium fluoride (TBAF) in dimethylformamide (DMF) (15 mL) was stirred at room temperature for 2 h. Excess solvent was removed, and the resulting residue was directly purified on silica-gel flash column chromatography with gradient DCM-ethanol (up to 15% ethanol) to afford 4NSG (4) as a white solid .The 4NSG was synthesized according to Fig. 1H NMR  δ 10.93 , 8.20 , 7.25 , 6.27 , 6.14 , 5.27 – 5.24 , 4.19 – 4.14 , 3.86 , 3.80 – 3.75 , 3.65 – 3.59 , 2.40 – 2.34 , 1.51 , 1.26 – 1.15 , 0.82 .13C NMR  δ 174.53, 163.33, 154.64, 145.16, 123.38, 96.34, 84.55, 81.47, 68.83, 59.23, 36.83, 31.72, 29.46, 29.44, 29.42, 29.40, 29.27, 29.13, 28.87, 24.78, 22.52, 14.38. MP = (145–146 °C), Rf value = 0.54 (100% ethyl-acetate). HPLC (H2O/ACN (90:10), % purity = 99.8%, RT = 14.3 min) MS (M + H)+  = 530.59, Molecular formula: C27H45F2N3O5•H2O, Mol wt: (4NSG) 547.68. Elemental Analysis: Calculated: C = 59.21; H = 8.65; N = 7.67; F = 6.94. Found: C = 59.27; H = 8.59; N = 7.51; F = 6.67.The metabolic stability of 4NSG was examined in the presence of PBS (pH 7.4) and human liver microsomes as previously described , 26. HumTo determine concentrations of 4NSG in PBS and human liver microsomes solutions, supernatants from PBS and human liver microsomes controls were spiked with aliquots of 0.5 μg/ml of 4NSG. The chromatographic analysis was performed according to the method described with minor modifications , 28. The4NSG-SLN formulation was prepared by cold homogenization technique using a high-shear homogenizer technique with slight modifications . BrieflyThe morphological examination of 4NSG-SLN was performed using high-resolution TEM. Three microliters (3 mL) sample was stained using 50 μL of ammonium molybdate solution (1% w/v) after adjusting the pH of the 4NSG-SLN suspension to 7.0 with 5 N sodium hydroxide. The stained sample was placed on copper grids, allowed to dry, and viewed by a Tecnai F-20 transmission electron microscope (Philips Co. Japan).The particle size, polydispersity (PDI), and zeta potential were measured using a Zeta Potential/Particle Sizer (NICOMP 380 ZLS). All measurements were performed in triplicate. The NICOMP 380 ZLS estimated the nanoparticles based on principles of dynamic light scattering (DLS).The stability of 4NSG-SLN was analyzed after incubation in PBS (pH = 7.4) alone (control) and PBS with 25% (v/v) FBS. Both solutions were maintained at 37 °C in a water bath for 7 days. NICOMP 380 ZLS particle sizer was used to measure the hydrodynamic diameter and PDI of 4NSG-SLN in both PBS alone and PBS with 25% (v/v) FBS for 7 consecutive days.The entrapment efficiency of 4NSG-SLN was determined by suspending 10 mg of lyophilized 4NSG-SLN in 2 mL of PBS (pH 7.4). The mixture was centrifuged at 6000 rpm for 5 min, and the supernatant was subjected to HPLC  equipped with an auto-sampler photodiode array (2998 UV/Vis) detector and pumps. Separation was performed using a reverse-phase column  with a flow rate of 1.0 ml/min and injection volume of 20 μL at ambient temperature at 268 nm. The EE was calculated based on the equation below:Cumulative in vitro drug release studies were performed as previously described , 31. Fre3 per well in 96-well plates in triplicates for each drug concentration level and incubated at 5% CO2 and a temperature of 37 °C [2, 37 °C) for 2–4 h [PCa primary cell were cultured with Dulbecco's modified Eagle medium (DMEM) with high glucose and L-glutamine and supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PenStrep) . Brieflyof 37 °C . At 70 -or 2–4 h . GloMax®3 for 24 h at 37 °C. The cells were treated with fluorescein isothiocyanate (FITC)-labeled SLN in the growth medium. After 3 h, FITC-SLN was removed, and the cells were gently washed twice with PBS. Afterward, 5 μg/ml of Hoechst dye was added for nuclear staining; the cells were fixed using 4% paraformaldehyde. The fixed cells were mounted and imaged using Leica SP2 Multiphoton system [PPCL-192 and PPCL-46 cells were grown in 6-well plates (with coverslips) at a cell density of 2 × 10n system .5 in 6-well plates and cultured in a growth medium until 70% confluence to determine cellular uptake of 4NSG-SLN. In place of 4NSG, cells were treated with FITC and (FITC)-labeled SLN for 24 h at 37 °C. After treatment, cells were washed thrice with PBS and detached using 0.25% trypsin- ethylene diamine tetraacetic acid (EDTA) solution. Trypsin was deactivated by adding a culture medium and centrifuged at 6,000 rpm (3820 rcf) for 5 min. Cells were resuspended in 500 μl PBS and fixed with 4% paraformaldehyde. The fixed cells were kept on ice until analysis using a Becton Dickinson (BD) Fluorescence-Activated Cell Sorting Canto Analyzer and a BD Fluorescence-Activated Cell Sorting Aria Cell Sorter (BD Biosciences) [PPCL-192 and PPCL-46 cells were seeded at a cell density of 1 × 10ciences) .2 culture flask at a density of 5 × 105 cells and cultured in DMEM medium supplemented with 2 mM L-glutamine, 10 mM HEPES, 10% FBS, and 1% penicillin/streptomycin. After the cells reached 75% confluency, they were exposed to varying concentrations of  for GemHCl and 4NSG-SLN. After 48 h exposure, treatment was terminated, cells harvested, and then re-plated onto 6-well plates at a density of 200, 500, and 1,000 cells per well and incubated with a growth medium. After the cells reached 75% confluence, the experiment was terminated by fixing and staining the plates with 0.5% crystal violet solution. The stained colonies (fifty per colony) were counted using a Jenco™ Stereomicroscope; plating efficiency (PE) and surviving fraction (SF) were calculated, and a graph of survival curve graph was generated [For colony assay, PPCL-192 and PPCL-46 cells were seeded into a T-25cmenerated .5 cells per well) were seeded in 6-well plates and cultured using DMEM medium supplemented with 10% FBS10 mM HEPES, 10% FBS, and 1% penicillin/streptomycin were seeded in 6-well plates and incubated at 37 °C. After the cells reached 75% confluence, they were exposed to different concentrations  of GemHCl and 4NSG-SLN. After 24 h exposure, cells were harvested, washed with PBS, centrifuged at 300 × g for 5 min, resuspended the pellets in 100 μL of PBS, and passed through a 28 5/8 needle to disperse them into single cells. Fixation and permeation of cells were performed using cold 70% ethanol while vortexing. Cells were then stored at − 20 °C overnight. The cells were resuspended in 0.1 mg/mL RNase, stained with 40 mg/mL PI, and the phase distribution was examined using flow cytometry .Cell cycle study was performed to determine the effect of 4NSG-SLN on the cell cycle; PPCL-192 and PPCL-46 cells  and then further incubated for 48 h. After serum starvation, the inserts were gently removed to generate a gap or "wound" between the two confluent layers of cells, and the monolayers were washed with experimental media. Afterward, cells were exposed to 5 μM concentration of GemHCl and 4NSG-SLN treatments. Images of the cells invading the wound were then captured after 48 h with a Nikon Ti Eclipse microscope.A cell migration assay was conducted to determine the effect of 4NSG-SLN on PPCL-192 and PPCL-46 cell motility. The Ibidi cell culture inserts were used to grow the cells into two confluent monolayers separated by a "wound" for this assay. The cells were seeded into 24-well plates at a cell density of 2.5 × 10Eight-week-old mice were obtained from The Jackson Laboratory ..The mice were housed in a virus-free, indoor, light- and temperature-controlled barrier environment and were provided ad libitum access to food and water. All procedures with mice were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines.This was approved by the Florida A & M University Animal Care and Use CommitteeThe implantation of surgical tumor tissue into immuno-compromised mice was described previously , 36. A vFor pharmacokinetic (PK) studies, normal healthy mice were grouped into control, GemHCl, and 4NSG-SLN. Mice were given a single bolus intravenous injection of 30 mg/kg of Gem , while control mice received normal saline (0.9% NaCl). After the injection, aliquots of blood samples were collected at predetermined time points . Blood samples collected were treated with 2 mL of an extraction solvent  , 38. TheFive hundred microliters (500 uL) of mobile phase solution was used to reconstitute the dried sample . The sample solution was centrifuged at 3,000 rpm for 15 min, supernatant collected and filtered, and samples analyzed for Gem using HPLC. The PK parameters were determined using PKSolver software .To determine the concentration of Gem, supernatant from mouse plasma was spiked with aliquots of 0.5 μg/ml of Gem. The chromatographic analysis was performed according to the method described with minor modifications , 28. Thehttps://home.histowiz.com/faq/). Samples were incubated overnight with primary antibodies , and immunohistochemical staining was done using rabbit anti-rat secondary  [Tissue samples from mice bearing tumors from Black and White PCa patients post treatments with GemHCl and 4NSG-SLN were excised and immediately washed with PBS, fixed  for 24 h, and transferred to 70% ethanol for histopathological analysis. Histology was performed by HistoWiz Inc. (Histowiz.com) according to manufacturer protocol. Samples were embedded in paraffin, and 4 μm sections were prepared , 41. Imm, 1:100) , 41. Bon3 were randomized into groups as control, GemHCl, and 4NSG-SLN (n = 5/group). Baseline tumor volume was established, and dosing initiation began on day 1 with intravenous administration of 30 mg/kg Gem (twice weekly for 3 weeks) with an equivalent dose of GemHCl and 4NSG-SLN [2)/2, where V is the volume (mm3), W(width) is the smaller of two perpendicular tumor axes, and the L (length) is the larger of two vertical axes. Mean tumor volume growth curves were calculated for each treatment group [Mice-bearing surgically implanted tumors obtained from Black and White patients with sizes of 70–100 mm4NSG-SLN . Once tunt group .2), followed by decapitation. CO2 flow to the chamber was adjusted to 3 L per minute for 2 to 3 min and observed each mouse for lack of respiration and faded eye color. The CO2 flow was maintained for a minimum of 1 min after respiration ceased and followed by decapitation with scissors.After the tumor efficacy studies, mice were sacrificed by carbon dioxide .Pharmacokinetic parameters were estimated using the software PKSolver. The difference between GemHCl and 4NSG-SLN treatment groups was analyzed using Student's t-test and considered significant at 1H and 13C NMR, and the results observed were consistent with reported data [1H NMR peaks were 10.88 ppm (-CO–NH), and 1.38 – 1.04 ppm (-CH2-)15, representing the amide linkage and long-chain methylene group contributed by stearic acid, respectively. The formed amide bond in 4NSG revealed a characteristic 13C NMR peak at 174.55 ppm, confirming the presence of an amide bond between stearic acid and Gem. Based on micro elemental analysis results, , the purity of 4NSG was found to be 99.8%, and this result is similar to our HPLC purity analysis of 4NSG which was 99.8%. Mass spectrometer analysis of 4NSG was performed, and Supplementary Fig. 4NSG was successfully synthesized based on the data obtained from NMR, micro elemental analysis, and mass spectrometer. The NMR spectra of 4NSG were observed using ted data   ranging from 82 ± 2.0 nm to 89 ± 1.8 92 nm from 1The cumulative in vitro drug release of 4NSG from 4NSG-SLN was determined for a 12 h period at 37 °C while maintaining sink condition. The rapid release of free 4NSG (Mol. weight of 547.68) from the dialysis bag (MCO of 3.5 kDa) was about 60% within the first 2 h and a further 20% increase in the next 2 h. No significant increase in free 4NSG release was observed after 4 h  for 24 h. Figure Cell migration assay examined cell mobility towards a uniform "wound" made in confluent monolayer culture of PPCL-192 and PPCL-46 cells. The number of cells that migrated towards the wound was evaluated at 5 µM after 48 h. The control (untreated) PPCL-192 and PPCL-46 cells, after 48 h, migrated towards the wound, covering almost the entire surface area of the Ibidi cell culture inserts Fig. . 4NSG-SLp-value of 0.0001. The AUC also showed a similar trend where 4NSG-SLN (86.2 ± 5.43 µg/mL*h) showed a threefold increase in bioavailability compared to GemHCl (28.2 ± 4.3 µg/mL*h) with a p-value of 0.0001. Since half-life is inversely proportional to plasma clearance, it was observed that an increase in the half-life resulted in a decrease in clearance for 4NSG-SLN (7.1 ± 1.1 mL/h) compared with an increase in clearance for GemHCl (21.3 ± 3.2 mL/h) with a p-value of 0.0001.The pharmacokinetic parameters of 4NSG-SLN were determined by administering a 20 mg/kg bolus intravenous injection in mice. An equivalent dose of GemHCl was also administered to mice using the same route to evaluate and compare their pharmacokinetic parameters. As indicated in Table 3 to 800 mm3) was observed in 4NSG-SLN treated mice bearing tumors from Black patients on day 11, indicating a significant tumor inhibition compared to GemHCl treatments which were within the range of (1100 mm3 to 1700 mm3). A similar trend was observed on days (13 through 25) for 4NSG-SLN treatments, which showed an overall increase in antitumor efficacy in individual mice bearing PCa tumor from Black patients, as indicated in Fig. 3) in 4NSG-SLN mice bearing White’s tumor compared with that of GemHCl treatments ranging from (1200 mm3 to 2200 mm3) confirms a similar trend observed in days (13 through 25) as shown in Fig. Tumor heterogeneity is evident in PCa patients. To mimic this in our PDX study, each mouse-bearing tumor from Black and White PCa patients was treated individually to assess the antitumor activity of 4NSG-SLN compared to GemHCl treatments for 25 days. The normalized tumor volume of range between , HER2 (ControlBT = 18.4 ± 0.6 ControlWT = 27.3 ± 2.3) and VEGFR . The impact of the 4NSG-SLN formulation was evaluated on EGFR, HER2, and VEGFR expressions in the mice's pancreatic tumor tissue. Figure BT = 14.2 ± 5.5, 4NSG-SLNWT = 5 ± 2.2), HER2 4NSG-SLNBT = 2.3 ± 1.2 4NSG-SLNWT = 3.6 ± 1.9) and VEGFR . There was a significant reduction in EGFR, HER2 and VEGFR expressions in both 4NSG-SLN treated mice bearing Black and White tumor expressions as observed in Fig. The expressions of EGFR, HER2, and VEGFR were evaluated in mice pancreatic tumor tissues using EGFR, HER2, and VEGFR staining. Positive membrane staining was observed in EGFR  preserve in part the intratumoral heterogeneity that exists in pancreatic cancer is the new approach adopted for screening chemotherapeutic agents. The expansion of PPCLs from PDXs has been successful in 100% of attempts. Therefore, there is a need to utilize primary cells such as PPCL-135 and PPCL-192, PPCL-46 PPCL-68 from patient-derived xenografts, demonstrating highly reproducible, predictive, and translational values , 43, 44,Before formulation and in vitro studies, the structure and purity of 4NSG were confirmed using NMR, micro-elemental analysis, HPLC, and mass spectrometry. The purity of the 4NSG obtained suggests that the synthesized compound used in this study was pure and was significantly devoid of any unwanted material .Human liver microsome was used to determine the in vitro metabolic stability of 4NSG. We found that 4NSG was very stable and would likely remain intact or unchanged in the system circulation compared to GemHCl as reported in literature , 47.Pharmaceutical nano-drug delivery systems have been employed to target anticancer drugs to tumors of interest and reduce unintended distribution and adverse effects on healthy cells. Nano-drug delivery systems enhance drug properties, such as increased entrapment and prolonged systemic circulation.This protects anticancer drugs from first-pass metabolism and enzymatic degradation. SLNs possess biocompatible and biodegradable properties, which shield encapsulated drugs from harsh conditions . Recent 4NSG-SLN treatments against PPCL-192, PPCL-135, PPCL-46, and PPCL-68 cultures showed a higher antiproliferative activity compared to GemHCl. This might be attributed to the exceptional characteristics of 4NSG-SLN due to the modification of the polar nature of GemHCl to moderately lipophilic through the conjugation of Gem to stearic acid. Additionally, Gem has been reported to rely heavily on nucleoside transporters (hENT1) to deliver and accumulate into PCa cells. Under-expression of these transporters has been demonstrated to confer resistance to PCa . Our resDuring this process, lysosomal enzymes catalyze the degradation of the SLN, and the degradation facilitates the release of 4NSG from the nanoparticles. In the lysosome, enzymes such as cathepsin B catalyze the hydrolysis of 4NSG into Gem. The released Gem is exported from the lysosome into an appropriate intracellular compartment in the cytoplasm by nucleoside transporters such as lysosome-specific hENT3. Gem present in the cytoplasm undergoes phosphorylation and is converted into its active metabolites, dFdCDP and dFdCTP. 4NSG can diffuse and be transported into tumor cells via passive diffusion. However, due to the highly lipophilic nature of 4NSG, the release of Gem from 4NSG after hydrolysis might occur outside the suitable intracellular compartment for efficient phosphorylation. This might result in the likelihood of Gem undergoing deamination by cytidine deaminase, considering that nucleotides usually do not enter cells in the form of long-chain fatty acid conjugates , 53,.The cellular uptake of 4NSG-SLN was evaluated using flow cytometry and confocal imaging. Flow cytometry and confocal studies indicated a tenfold increase in geometric fluorescent intensity in FITC-SLN compared to FITC-exposed cells. This suggests that the SLN could entrap a higher payload of 4NSG, which confirms the increase in the intensity of FITC-SLN compared to FITC alone.The clonogenic assay measures the ability of the cells to proliferate and metastasize in forming colonies of at least 50 or more cells with a critical metric of cell viability . The datThe regulation of cell cycle progression in cancer cells is considered a potentially effective strategy for controlling tumor growth . Most caTherefore, 4NSG-SLN may have triggered apoptosis in the PPCL-192 and PPCL-46 cells at 20μΜ concentration which confirms the least percent cell population accumulated studies' G2/M phases  conjugation of Gem to stearic acid may have imparted some degree of lipophilicity to 4NSG, which may have facilitated its delivery to cancer cells, and iii) encapsulation of 4NSG into SLN delivery system protected 4NSG and increase Gem payload.Antitumor activity of 4NSG-SLN conducted over 25 days exhibited significant tumor growth inhibition compared with GemHCl in both Black’s and White’s tumors. The SLN may play an excellent role in encapsulating a higher payload of 4NSG. This led to high accumulation in the tumor site to cause a substantial tumor inhibition observed.in mice bearing Black’s and White’s tumors throughout the study period. Some critical factors could be used to explain the extraordinary tumor efficacy of 4NSG-SLN: i) absence of free NHIn this study, we demonstrated that 4NSG enhanced the therapeutic efficacy of Gem by improving its plasma metabolic stability and uptake into PCa cells. Chemical modification of Gem into 4NSG followed by formulation into 4NSG-SLN prolonged its systemic circulation and improved the bioavailability of Gem. Altogether, 4NSG-SLN showed a significant tumor inhibition in mice bearing Black and White patients’ pancreatic tumors compared with the GemHCl-treated mice bearing Black and White pancreatic tumors. Except for Black tumor sensitivity to 4NSG in the first week of treatment, no significant difference in 4NSG sensitivity was observed between Black and White tumors. Due to biological or genetic factors, an increased number of Black and White tumors may provide a definite difference in PCa status in our future studies.Additional file 1: Supplementary Fig 1. A (SF 1A). HPLC spectrum for detecting the purity of 4NSG at 98.8% with a wavelength of 254 nm. B (SF 1B). Nuclear magnetic resonance spectrum for 4NSG. (a) 4NSG proton nuclear magnetic resonance (1H NMR) peaks at 10.88 ppm (-CO-NH) and 1.38–1.04 ppm (-CH2)15 representing the amide linkage and long-chain methylene group contributed by stearic acid respectively. (b) amide carbonyl carbon in 4NSG displays a characteristic C-13 NMR peak at 174.55 ppm. C (SF 1C). Mass spectrum for 4NSG at 530.59 m/z.Additional file 2: Supplementary Fig 2. (SF2 ) (a) Dynamic light scattering (DLS) graph showing the hydrodynamic particle size distribution of freshly prepared SLN with a mean diameter of 35 nm, (b) Dynamic light scattering (DLS) graph showing the hydrodynamic particle size distribution of freshlyprepared SLN with a mean diameter of 82 nm, (c) Graph of zeta potential distribution for 4NSG-SLN.


BiVO4 exists in four polymorphs: orthorhombic, zircon-tetragonal, monoclinic, and scheelite-tetragonal structures. In these crystal structures, V is coordinated by four O atoms in tetrahedral coordination and each Bi is coordinated to eight O atoms from eight different VO4 tetrahedral units. The synthesis and characterization of doped bismuth vanadate with Ca and Cr are studied using gel methods , which are compared with the ceramic route by means of the UV–vis–NIR spectroscopy of diffuse reflectance studies, band gap measurement, photocatalytic activity on Orange II and its relation with the chemical crystallography analyzed by the XRD, SEM-EDX and TEM-SAD techniques. The preparation of bismuth vanadate-based materials doped with calcium or chromium with various functionalities is addressed (a) as pigments for paints and for glazes in the chrome samples, with a color gradation from turquoise to black, depending on whether the synthesis is by the conventional ceramic route or by means of citrate gels, respectively; (b) with high NIR reflectance values that make them suitable as fresh pigments, to refresh the walls or roofs of buildings colored with them; and (c) with photocatalytic activity.Clinobisvanite (monoclinic scheelite BiVO Although orthorhombic is the most common phase in nature , it has not been synthesized in the laboratory. The low-temperature synthesis of BiVO4 produces the zircon-tetragonal phase with a band gap of 2.9 eV, which, at 528 K, transforms into the monoclinic phase, reversible to tetragonal by adjusting the temperature. In these crystal structures, V is coordinated by four O atoms in a tetrahedral coordination and each Bi is coordinated to eight O atoms [8 polyhedron known as bisdisphenoid or dodecadeltahedron is rather familiar in this case because all polymorphs of described BiVO4 show this typical coordination in zircon and scheelite; this polyhedron has 12 triangular faces, 18 edges and, of course, 8 vertices with symmetry D2d in Schönflies notation [Monoclinic BiVOironment . BiVO4 e4 I/b is a dnotation .2)-based visible-light-driven photocatalysts, monoclinic BiVO4 has proved to be an excellent material for photocatalytic water splitting and the photocatalytic degradation of organic compounds [Among non-titania (TiOompounds .4 is not classified as hazardous and is used to replace the highly toxic yellow cadmium sulfoselenide in ecofriendly paints applications [6H4(OH)CO2)3)), an antacid and anti-diarrheal agent [BiVOications . In geneications  and or tal agent .4 can be stabilized in different crystal structures, yet high PEC efficiency is found only for the n-type-doped monoclinic phase. The theoretical description of the monoclinic polymorph is difficult because of spontaneous transformation to a tetragonal phase. The cause of instability of the monoclinic phase which spontaneously transforms to a tetragonal phase has not solved yet. Laraib et al. [4 because m-BiVO4 is related to the higher photoelectrochemical activity.BiVOb et al.  suggest 4”, there are three entries (two in WOS and another one in Google): one for visible-light-activated (VLA) photocatalysis applied to the degradation of pharmaceutical contaminants of emerging concern and two for photoanodes for water splitting, all based in the semiconductor characteristics of Ca-BiVO4 solid solutions. Fatwa et al. [4 photoelectrodes, and the resulting Ca-doped BiVO4 photoelectrodes show anodic photocurrents with an enhanced carrier separation efficiency. BiVO4 and Ca-BiVO4 show indirect semiconductor behavior with band gap measured by a Tauc plot of 2.48 and 2.44 eV, respectively. Li et al. [IIxBi1−xV1−xMoxO4  by the ceramic method; the samples x = 0.1 in the cases of samples containing calcium and strontium afford the highest water-splitting performance.A simple review of the literature about the studied systems has been carried out analyzing the entries in WOS (Web of Science) and Google Academics. For the topic “calcium-doped BiVOa et al.  introduci et al.  prepare 4”, twelve entries are detected—nine in WOS and two in Google. Seven are related to different photocatalytic systems applied to the photoreduction of chromium (VI) in wastewater and the other four to photoelectrochemical water splitting. The Cr-doped BiVO4 are synthesized using chromates via liquid–solid state reaction obtaining Cr(VI) solid solutions in BiVO4, where Cr(VI) replaces V(V). Krysiak et al. [4 using a liquid–solid state reaction in ethanol media by means of multicomposite catalysts containing nickel, iron, and chromium introduced as nitrates. The formed catalyst particles were not uniformly incorporated into the Mo-BiVO4 structure, but rather creating agglomerates. This suggests phase segregation, which may be beneficial for photoelectrochemical activity. Okuno et al. [6+-BiVO4 solid solutions from Bi(NO3)3.5H2O, V2O5 and CaCrO4 in HNO3. The resulting solution was stirred for 26–168 h in the dark and the resulting precipitate was washed and dried at 80 °C for 5 h. The single phase of monoclinic scheelite BiVO4 was obtained with up to 2% Cr doping while mixtures of monoclinic scheelite and tetragonal-zircon BiVO4 were obtained for samples with larger amounts of doping (3–5%) even with a prolonged reaction time of up to 4 weeks.In the case of “chromium-doped BiVOk et al.  studied o et al.  prepared4 applies to the photoelectrochemical splitting of water and uses solid-state liquid reactions in organic media to preserve high valence ions such as Cr(VI). Therefore, it is not applicable for the synthesis of ceramic pigments that need high temperatures to be used in ceramic glazes and earthenware (1000–1300 °C) in construction applications.Therefore, the literature related to Ca- and Cr-doped BiVOThis work addresses the synthesis and characterization of bismuth vanadate doped with calcium or chromium with various functionalities (a) as pigments for paints and enamels; (b) with high NIR reflectance values that make them suitable as cool pigments, to refresh the walls or floors of buildings colored with them; and (c) with photocatalytic activity.The synthesis methodology will be based on the ceramic method CE, the ammonia coprecipitation method CO and the metal–organic decomposition method MOD.2O3, α-Bi2O3 and NH4VO3 99.9 wt% supplied by Sigma-Aldrich. These precursors with a particle size between 0.3 and 5 µm were mechanically homogenized in an electric grinder  for 5 min and the mixture fired at the corresponding temperature and soaking time.Ceramic samples (CE) were synthesized from Cr3)3.9H2O, NH4VO3 (previously dissolved in HNO3 30 wt%) and Bi(NO3)3.5H2O . For 5 g of the product, these precursors were dissolved in 200 mL of water, then citric acid is added with a molar ratio of Bi:Acid = 1:x . This solution was continuously stirred at 70 °C and ammonia 17.5 wt% was dropped until gelation occurred at approximatively pH 7.5. The gel was dried at 110 °C and fired at the corresponding temperature and soaking time .CO and MOD samples were synthesized from Cr.X-ray diffraction (XRD) was carried out on a Siemens D5000 diffractometer  using Cu KL*a*b* and C*h* color parameters of glazed samples were measured following the CIE-L*a*b*  colorimetric method using a X-Rite SP60 spectrometer, with standard lighting D65 and a 10° observer . L* measWe can point out that the optimal chroma is obtained when lightness L* (measured by the L*a*b* method) is high for yellow (80–90), cyan (75–85) and green (70–80), middle for magenta (50–60) and red (45–55) and low for blue (25–35) hues.UV–vis–NIR spectra of fired powder and the applications of the pigments were carried out by a Jasco V670 spectrometer  through the diffuse reflectance technique, measuring absorbance (A in arbitrary units) or reflectance (R(%)) through the Kubelka–Munk transformation model. A band gap energy of semiconductors was calculated by a Tauc plot from the UV–vis–NIR diffuse reflectance spectra .R or total solar reflectance, RNIR or solar reflectance in the NIR and RVis solar reflectance in the visible spectrum are evaluated from UV–vis–NIR spectra, through the diffuse reflectance technique, as the integral of the measured spectral reflectance and the solar irradiance divided by the integral of the solar irradiance in the range of 350–2500 nm for R, 700–2500 nm for RNIR and 350–700 nm for RVis as in the Equation (3):r(λ) is the spectral reflectance (Wm−2) measured from UV–vis–NIR spectra and (b) i(λ) is the standard solar irradiation (Wm−2 nm−1) according to the American Society for Testing and Materials (ASTM) Standard G173-03.The solar reflectance, Microstructure characterization of powders was carried out by scanning electron microscopy (SEM) using a JEOL 7001F electron microscope  and transmission electron microscopy (TEM) using a HITACHI electron microscope and selected area electron diffraction (SADP) .−5 M of Orange II in pH 7.42 phosphate buffer media (NaH2PO4·H2O 3.31 g and Na2HPO4·7H2O 33.77 g solved in l.000 mL of water). The degradation of Orange II in buffer media was followed by colorimetry at λ = 485. The UV irradiation source was a mercury lamp of 125 W emitting in the range 254–365 nm. The suspension was first stirred in the dark for 15 min to reach the equilibrium sorption of the dye. Aliquot samples were taken every 15 min to measure the change in the dye concentration. Control experiments with Orange II solution and without catalyst, were conducted before the photocatalytic experiments (CONTROL). Commercial anatase P25 from Degussa was used as a reference to compare its photocatalytic activity with the studied samples [The photocatalytic tests were performed using a dispersion of 500 mg/L of powder added to a solution 0.6·10 samples .1/2 and the correlation of kinetic data R2.The photodegradation curves were analyzed following the Langmuir–Hinshelwood model  measurin2(62.3), B2O3(9.1), Al2O3(10.3), CaO(5.9), K2O(9.3), ZnO(3.1), ZrO2(0.1)) supplied by Alfarben S.A., with a maturation point at 1050 °C that devitrifies zircon. The mixture of the frit and the pigment were fired applying a standard firing cycle used in the ceramic tile industry, maintaining the maximum of temperature (1050 °C) for 5 min.In order to analyze the pigmenting capacity of powders as ceramic pigments, the powders were 3 wt% mixed with a double-firing frit (composition in oxides (wt%); SiO3+(VIII) = 1.31 Å, Ca2+(VIII) = 1.26 Å, Cr3+(VIII) = 0.7 Å) and the crystalline field over the d orbitals goes down; therefore, the absorption bands shift to higher wavelengths. In short, in this mechanism, there is a cooperative transition between the chromophore and the replaced ion that modifies the crystalline field over the chromophore ion.A cooperative transition (COT) mechanism is assumed . In this3+ by the smaller Ca2+ or Cr3+ should relax the dopant ions, which shifts its absorption to higher wavelengths, but aliovalent replacement with Ca2+ probably increases the concentration of charge-carrying mobile defects of oxygen [In this case, the replacement of Bif oxygen ,18.xBi1−x)VO4, x = 0, 0.1, 0.2, 0.4 (600 °C/3 h): (a) aspect by binocular lens (×40), and its L*a*b*, and (b) 3 wt% washed samples glazed in a double-firing frit (1050 °C). The entrance of Ca2+ replacing Bi3+ is associated with a slight decrease in color intensity . The pigments are dissolved, unstabilized in the glaze, and do not produce coloration (loration b.xBi1−x)VO4, x = 0.1, 0.2, 0.4 (600 °C/3 h), showing a transference charge band centered at 400 nm with a typical semiconductor performance. xBi1−x)VO4 (600 °C/3 h) samples: the band gap measured from UV–vis–NIR spectra by the Tauc procedure (4 (x = 0) to 2.31 eV for the x = 0.4 doped sample (10). Thereby, NIR reflectance also increases continuously with x: from 53% for pure BiVO4 to 62% for the x = 0.4 sample, improving the cool performance of BiVO4.rocedure  indicate1−xCaxVO4 ceramic samples show clinobisvanite (V) as a single phase, except the x = 0.4 sample, which shows residual peaks of CaV2O6 (see 2+ in Bi1−xCaxVO4−x/2 is lower than x = 0.4 and stabilizes the monoclinic polymorph (11).All XRD of these BiV2O6 see . TherefoxBi1−x)VO4, x = 0, 0.1, 0.2, 0.4 fired at 600 °C/3 h). 1−xCrxVO4 ceramic samples: all samples show clinobisvanite (V on figure) as majoritarian phase with residual peaks associated with hexagonal Cr2O3 (C) and Scherbinite V2O5 (S), which slightly increase in intensity with the amount of doping agent x (best observed in the highly doped sample (x = 0.4)).When washed powders were 3 wt% glazed in a double-firing frit (1050 °C) b an intexBi1−x)VO4, x = 0.1, 0.2, 0.4 (600 °C/3 h) powders (see spectra on 3+ (3d3) in the dodecadeltahedral position (D2d symmetry group), with a multiband at 220–520 nm integrated almost for the overlap of bands centered at 270, 380 and 480 nm, respectively, and a shoulder at 750 nm (that confers a semiconductor appearance to the spectrum) with bands at 680 and a shoulder at 760 nm. The electronic configuration for Cr3+(d3) may be described as d x2−y21d z21 dxz,yz1dxzdxy [xBi1−x)VO4 (600 °C/3 h) samples: the band gap measured from UV–vis–NIR spectra by the Tauc procedure indicates that the semiconductor shows a direct performance, decreasing its band gap slowly from 2.25 eV for pure BiVO4 (x = 0) to 2.13 eV for the x = 0.4 doped sample. Likewise, NIR reflectance also decreases continuously with x: from 53% for pure BiVO4 to 41% for the x = 0.4 sample, reducing the cool performance of BiVO4.The UV–vis–NIR spectra of (Crz1dxzdxy . Table 1xBi1−x)VO4, x = 0.1, 0.2, 0.4 (600 °C/3 h) glazed samples. It is detected as an intense band centered at 270 nm to the charge transfer band of the frit used in the glaze, and relatively intense bands in the visible range at 380, 500 and 620 with a shoulder at 720 nm; a minimum of absorbance at 520 nm is detected, indeed a maximum of light reflected, that explains the turquoise color of the sample. These bands can be considered the same as that of the powders shifting to lower wavelengths.xBi1−x)VO4, x = 0, 0.1, 0.2, 0.4 (600 °C/3 h) glazed samples showing a direct type semiconductor behavior. For the pure BiVO4 (x = 0) that unstabilizes in the glaze and produces white color associated with the glaze , 3 wt% 3NaF.2MgF2.Li2CO3 (min 2) and 2 wt% CaCO3.KCl +1 wt% NaF.Na4B2O7 (min 3). The DTA and TG analyses of the employed mineralizers  is greenish  but the turquoise shade obtained in glazed samples is similar to the unmineralized sample, the middle mineralizer 2 produces green shades in both powder and glazed samples, and the higher-temperature mineralizer (min 3) is also greenish in powder but shows a high turquoise intensity in glazes (L* decreases and negative b* increases). The addition of mineralizers only shows a significant improvement of the turquoise shade of the glazed sample with mineralizer 3 with a near thermal activity (580 °C) to the firing employed temperature (600 °C) (20).4 and residual peaks of S (scherbinite V2O5) and C . In the case of mineralizer 2, C  and very weak peaks of W (CrVO4) are detected; finally, for mineralizer 3 only residual peaks of W (CrVO4) are detected. Therefore, the relative high-temperature mineralizer 3 shows the cleaner diffractogram  and the best turquoise result in the glaze.0.4Bi0.6VO4 powders and glazed mineralized samples, respectively. As described above, powders show the optical absorption bands associated with Cr3+ (3d3) in the dodecadeltahedral position but the absorbance is higher in mineralized samples; and in the case of min 3, the shoulder at 680 nm practically disappears. Likewise, glazed samples show an intense band centered at 300 nm due to the charge transfer band of the frit used in the glaze and relatively intense bands in the visible range at 500, 610 and the shoulder at 680 nm; a minimum of absorbance at 500 nm is detected; indeed a maximum of light reflected, which explains the turquoise color of the sample. The band gap of powders (measured by the Tauc method) decreases for mineralized samples; 2.11 eV for min 1 and 2.09 eV for min 2 and min 3  (R = 43–44)  . On the = 43–44) , in agreThe charred powders at 500 °C/1 h I are of 4 are detected. At 600 °C, as in the CE sample, the CO and MOD samples show clinobisvanite m-BiVO4 with residual peaks associated with S (scherbinite V2O5) and C ; in the CO and MOD samples, the intensity of residual phases slightly decreases.3+ (3d3) in the dodecadeltahedral position (D2d symmetry group) are detected in the case of the yellow powder . For the greenish powder , the spectrum is similar, but the absorbance in the 580–1500 nm range increases. CE and mineralized yellow powders produce turquoise colors in the glaze and greenish CO and MOD powders give grey-black colors in the glaze. 0.4Bi0.6VO4 composition: (a) CITRIC MOD glazed samples 500 °C/1 h; (b) CE and CITRIC x = 2 samples 600 °C/3 h. (including sample CE). Bands at 270, 320,420, 580 and 670 nm can be detected, showing a shift at lower wavelengths relative to ceramic samples and a significant increase in absorption to approximately 500 nm associated with the darkening of glazes. The direct band gap of the semiconductors measured from Tauc plots, as shown in Vis and RNIR reflectance of the powders decrease slightly in CO and MOD samples associated with greenish hue (lower L*)  . On the her RNIR , in agre0.4Bi0.6VO4) fired at 600 °C/3 h (CE and CITRATE (x = 2) samples) with the Langmuir–Hinshelwood kinetics parameters t1/2  which for the commercial reference P25 of Degussa and for the control (without powder addition) are t1/2/R2= 25 min/0.898 and 497 min/0.945, respectively. The ceramic samples show an interesting degradation half-time of 85 min that increases to 150 min for the citrate sample (lower than 497 min for the simple photolysis of the CONTROL test).0.4Bi0.6VO4 composition: the CE powder, which produces a turquoise color on glazes, shows well-developed prismatic crystals of approximately 3 µm in length together with other irregular particles of 0.5 µm in size. The coprecipitated sample CO, which produces a grey color on glazes, shows very fine nanoparticles, forming agglomerates of 3–15 µm of size, finally MOD x = 0.25, which produces a grey color on glazes, shows microcrystals between 0.5 and 1 µm, forming big agglomerates of 10–20 µm. The decrease in the active surface due to the agglomeration of the fine particles of BiVO4 of the CO and MOD methods, observed by SEM, is probably associated with the decrease in the degradation half-time detected in the photodegradation test of these samples (12).4 are detected.Doped bismuth vanadate was synthesized using gel methods  and compared with the ceramic route.2+ in Bi1−xCaxVO4−x/2 is lower than x = 0.4, in the case of Cr3+ fired at 600 °C, and residual Cr2O3 and V2O5 always remained, indicating an incomplete solid solution of chromium in clinobisvanite (m-BiVO4), but both cations stabilize the monoclinic polymorph. NIR reflectance increases continuously with Ca doping from 53% for pure BiVO4 to 62% (Eg = 2.31 eV) for Ca0.4Bi0.6VO4 samples, improving the cool performance of BiVO4; conversely, the entrance of chromium decreases the NIR reflectance of clinobisvanite. The band gap of clinobisvanite increases for Ca-doped samples and decreases for Cr-doped samples in agreement with NIR reflectance evolution.The limit of the solid solution of Ca0.4Bi0.6VO4 sample (L*a*b* = 53.8/−5.8/−8.9) that maintains NIR reflectance at approximately 67% (band gap 2.13 eV). The use of mineralizer 3 (2 wt% CaCO3.KCl +1 wt% NaF.Na4B2O7) with a near thermal activity (580 °C) to the employed firing temperature (600 °C) produces a significant improvement in the turquoise shade of the glazed sample (L*a*b* = 49.6/−9.4/−9.4) with similar NIR reflectance (66%) and band gap (2.09 eV).The yellow samples doped with Ca do not produce coloration in glazes but the yellow–green Cr-doped samples produce interesting turquoise shades in a double-firing frit (1050 °C) resulting in the optimum turquoise of the CrCr-doped samples prepared by gel methods (CO and citrate MOD routes) produce yellow–green powders that darken with the amount of chromium, decreasing NIR reflectance and increasing the band gap. The corresponding glazed samples produce black shades with high NIR reflectance (69–71%) associated with a shift in the absorbance bands of Cr probably in dodecadeltahedral site to lower wavelengths relative to the ceramic sample and a significant increase in absorption at approximately 500 nm associated with the darkening of glazes.The Cr-doped ceramic samples show an interesting degradation half time of 85 min that increases to 150 min for the citrate sample (lower than the 497 min for the simple photolysis of the CONTROL test).4 pigments have been prepared for the first time for paints, and for glazes in the case of chromium, producing colors from turquoise to black depending on whether the synthesis is a conventional ceramic route or through citrate gels, showing a high NIR reflectance that makes them suitable as cool pigments, to refresh the walls and roofs of buildings colored with them, and gives them photocatalytic activity.To the best of our knowledge, Ca- or Cr-doped polyfunctional BiVO


People are widely exposed to polycyclic aromatic hydrocarbons, like benzo[a]pyrene (BaP). Prior studies showed that prenatal exposure to BaP depletes germ cells in ovaries, causing earlier onset of ovarian senescence post-natally; developing testes were affected at higher doses than ovaries. Our primary objective was to determine if prenatal BaP exposure results in transgenerational effects on ovaries and testes. We orally dosed pregnant germ cell-specific EGFP-expressing mice (F0) with 0.033, 0.2, or 2 mg/kg-day BaP or vehicle from embryonic day (E) 6.5–11.5 (F1 offspring) or E6.5–15.5 (F2 and F3). Ovarian germ cells at E13.5 and follicle numbers at postnatal day 21 were significantly decreased in F3 females at all doses of BaP; testicular germ cell numbers were not affected. E13.5 germ cell RNA-sequencing revealed significantly increased expression of male-specific genes in female germ cells across generations and BaP doses. Next, we compared the ovarian effects of 2 mg/kg-day BaP dosing to wild type C57BL/6J F0 dams from E6.5–11.5 or E12.5–17.5. We observed no effects on F3 ovarian follicle numbers with either of the shorter dosing windows. Our results demonstrate that F0 BaP exposure from E6.5–15.5 decreased the number of and partially disrupted transcriptomic sexual identity of female germ cells transgenerationally. People are exposed to PAHs largely via inhalation of particulate matter air pollution and tobacco smoke and via consumption of grilled or smoked foods3. Human biomonitoring data demonstrate that essentially all people are exposed to PAHs4. The PAH benzo[a]pyrene (BaP) is composed of five fused aromatic rings1. BaP, like other PAHs, undergoes metabolism to reactive metabolites, which are largely responsible for its toxicity5. Metabolic pathways include oxidation by cytochrome P450s, yielding epoxides, followed by epoxide hydrolase-mediated conversion to dihydrodiols. The latter can undergo further metabolism to highly reactive diol epoxides and o-quinones, which react with DNA forming bulky adducts8. The o-quinones can undergo redox cycling, resulting in formation of reactive oxygen species8. Another pathway, in which BaP acts as a reducing substrate for peroxidases, yields BaP radical cations, which are also DNA reactive9. Several BaP metabolites are aryl hydrocarbon receptor agonists and others are estrogen receptor agonists11. Thus exposure to BaP may result in toxic effects by inducing DNA damage or by activating arylhydrocarbon or estrogen receptors to initiate aberrant signaling.Polycyclic aromatic hydrocarbons (PAHs) are products of incomplete combustion of fossil fuels, wood, tobacco, food and other organic materials12. Females are born with a finite number of oocytes, which become surrounded by specialized somatic cells called granulosa cells, forming primordial follicles during the first postnatal week in mice and during the second trimester in humans13. Any exposure that decreases the size of the primordial follicle pool, often referred to as the ovarian reserve, will result in early onset of reproductive senescence in females. In contrast, males possess germline stem cells throughout life. Ovarian follicle numbers and testicular spermatid numbers were dose-dependently decreased following in utero exposure to 2 and 10 mg/kg-day BaP from E6.5–15.5, with significant effects observed at the lower dose in the F1 female offspring, but only at the higher dose in the F1 males15. Embryos deficient in the antioxidant and Phase 2 biotransformation cofactor glutathione were more sensitive to the in utero gonadal toxicity of BaP than wild type littermates15. BaP17 and another PAH, 9,10-dimethylbenz[a]anthracene19, induced apoptosis in germ cells of cultured E13.5 ovaries, but not E13.5 testes16, via BAX mediated caspase activation. Consistent with greater sensitivity of the developing ovary than the developing testis to BaP, the E13.5 ovarian transcriptome was significantly affected by BaP exposure, while the testicular E13.5 transcriptome was not significantly affected20.In utero exposure to BaP dose-dependently decreased fertility of male and female offspring, with complete sterility observed in both sexes with doses of 40 mg/kg-day and higher on embryonic days (E) 7–1624. Similarly, exposures of males to chemical toxicants have been shown to cause heritable effects in their offspring24. Effects are called multigenerational if they occur in the exposed F1 and F2 offspring of exposed F0 pregnant females or in the F1 offspring of exposed F0 males. They are considered transgenerational if they occur in the F3 or later generations descended from exposed F0 females or in the F2 and later generations descended from exposed F0 males. Several epigenetic mechanisms have been proposed for transgenerational inheritance including alterations in DNA methylation, histone modifications, and noncoding RNAs25. Mohamed et al.26 demonstrated F2 effects of F0 male dosing with 0, 1, or 10 mg/kg-day BaP orally for 6 wk prior to mating to an untreated female, followed by mating of F1 and F2 males with unexposed females to generate F2 and F3 males. They reported significantly decreased numbers of seminiferous tubules having elongated spermatids, as well as decreased epididymal sperm counts in F0, F1, and F2 males, and no effects in F3 males26. To our knowledge this is the only published study that investigated transgenerational effects of any PAH.In recent years there has been growing concern that in utero exposure to chemical toxicants can cause adverse health outcomes not only in the developing embryo of an exposed F0 pregnant female (F1 offspring), but also in their F2 daughters exposed as germ cells in the developing F1 female and their unexposed F3 grand-daughters27, proliferate and begin migrating along the hindgut mesentery to the gonadal ridges, where they continue to proliferate until beginning to enter meiosis in the female at E13.5  and undergoing mitotic arrest in the male at E13.528. E13.5 is also the time point during development when the germ cell DNA is maximally demethylated in both sexes31. The male E13 and E16 primordial germ cell transcriptomes were reportedly altered in a transgenerational manner after in utero exposure to the fungicide vinclozolin33. No prior studies of which we are aware have reported on the effects of in utero exposure to any environmental toxicant on the ovarian PGC transcriptome.In the mouse, the primordial germ cell (PGC) precursors form in the proximal epiblast around E6.0–6.5, become specified to PGCs around E7.25. Finally, we hypothesized that the mitotic window of ovarian development is more sensitive than the predominantly meiotic window of ovarian development to direct and transgenerational effects of in utero exposure to BaP.We hypothesized that in utero exposure to BaP results in diminished ovarian reserve not only in the F1 female offspring, but also in the F2 and F3 females and that this is associated with transgenerational alterations in the E13.5 ovarian PGC transcriptomes. Further, we hypothesized that males are less sensitive to the transgenerational gonadal effects of in utero exposure to BaP than females14. The POU transcription factor OCT4, also known as POU5F1, is expressed in proliferating PGCs, is downregulated when oocytes initiate meiosis, and is expressed again in oocytes from the primordial follicle stage onward34. We utilized mice expressing Enhanced Green Fluorescent Protein under control of Oct4 promoter and enhancers (OG2 mice)35 as a model system to investigate the effects of in utero exposure to BaP during critical windows of gonadal development, from E6.5 to E17.5, on germ cell numbers and on the E13.5 PGC transcriptomes in F1, F2, and F3 generations. We orally dosed pregnant F0 OG2 dams with 0.033, 0.2, or 2 mg/kg-day from E6.5–11.5 for collection of F1 ovaries and testes at E13.5 and from E6.5–15.5 for collection of F3 gonads at E13.5 and PND21. F0 exposure to 2 mg/kg-day BaP from E6.5–11.5 resulted in a statistically significant decrease in ovarian germ cell numbers in F1 females exposed prenatally to 2 mg/kg-day BaP at E13.5 , but not in F3 E13.5 males .We previously reported that oral dosing of pregnant F0 dams with 2 or 10 mg/kg-day BaP from E6.5–15.5 significantly and dose-dependently decreased ovarian reserve in F1 offspring36. However, in contrast to the results with F0 BaP dosing from E6.5–15.5, we observed no statistically significant effects of F0 BaP treatment on ovarian follicle numbers in the adult F2 or pubertal F3 females descended from F0 mice dosed during either of the shorter mitotic or meiotic developmental windows  with dosing from E12.5–17.5 (meiotic window) on ovarian follicle numbers in F2 and F3 females. We previously reported statistically significantly decreased primordial, primary, and secondary follicle numbers in BaP-exposed F1 females from this experiment at puberty, defined as first vaginal estrus, with no effect of exposure windowGclm, the modifier subunit of the rate-limiting enzyme in glutathione synthesis), we found that primordial, primary, and secondary follicles per ovary were significantly decreased in 4–5 month old F2 Gclm+/− mice descended from Gclm+/− F0 dams dosed from E6.5–15.5 with 2 mg/kg-day BaP compared to F2 mice descended from control F0 dams  in the E13.5 ovaries and none in the E13.5 testes20. In the present study we attempted to examine whether in utero exposure of mouse embryos to BaP affects sex differentiation of primordial germ cells (PGCs). In mice, PGCs arrive at the gonadal ridge E10.537, where they proliferate until entering meiosis in females starting at E13.5 , while in males they proliferate, migrate to the forming seminiferous cords 38, and undergo mitotic arrest shortly before birth38. Herein we refer to E13.5 germ cells as PGCs in both sexes as a shorthand. To collect E13.5 mouse PGCs, we took advantage of the OG2 transgenic mice35, which express EGFP from the germline-specific Pou5f1 promoter, as we previously described39. OG2 homozygous females were timed-mated with OG2 homozygous males. At E13.5, when PGCs are settled in the embryonic gonads and have initiated sexual differentiation as dictated by the surrounding gonadal somatic cells40, EGFP-expressing PGCs  taking a similar approach and the same set of DEG criteria as ours but with limited numbers of biological replicates (N = 2 for each sex). Secondly, we identified 3100 genes whose expression counts exceeded 100 in any E13.5 PGCs in any generations of embryos (F1–F3) with or without F0 exposure to BaP  regardless of the sample size.After the list of sex-specific DEGs, exposure-specific DEGs, and significantly expressed genes had been generated, we attempted to integrate the sex-specific DEGs and the exposure-specific DEGs. To prepare for this analysis, we performed permutation tests to establish the probabilities of accidentally observing sex-specific DEGs in a randomly sampled list of significantly expressed genes Fig. . For exaWe then determined numbers of the sex-specific DEGs in varying numbers of the statistically most significant exposure-specific DEGs and calculated the percentages of sex-specific DEGs per 1–200 exposure-specific DEGs Fig. A,C–F. WhAs noted above, using the stringent criterion of FDR p-value, we observed few statistically significant DEGs when comparing BaP-exposed to vehicle-exposed PGCs in any generation. Specifically, by this criterion, we observed 0, 1, and 7 DEGs, respectively, in the F1 0.033. 0.2, and 2.0 mg/kg-day BaP-exposed compared to oil exposed females; none in the F2 BaP-exposed compared to oil-exposed females, and 25, 6, and 3 DEGs, respectively, in the F3 0.033. 0.2, and 2.0 mg/kg-day ancestrally BaP-exposed compared to oil exposed females Tables , and S6.42. Specifically, we used lists of DEGs, which were 1.5-fold or more up or down-regulated and had unadjusted p-values < 0.05, as the target genes against the background list of all 15,423 expressed genes. We then used Revigo to remove redundant GO terms43.To better understand which biological processes may be driving the transgenerational sex disturbances, we used less stringent criteria for inclusion of DEGs in Gene Ontology (GO) analyses using GOrillaNo GO terms that were statistically significant by FDR p-value were identified by GOrilla in females from the F1 2 mg/kg-day or 0.033 mg/kg-day, any F2 dose groups, or F3 2 mg/kg-day and 0.2 mg/kg-day groups compared to oil controls.For F1 0.2 mg/kg-day exposed females compared to oil controls, GOrilla identified 21 GO terms, of which 18 were non-redundant and two had statistically significant FDR p-values (inflammatory response and defense response). For the F3 0.033 mg/kg-day exposed females compared to control females, GOrilla identified 157 GO terms, of which 108 were non-redundant and 88 had statistically significant FDR p-values and of which many related to immune, inflammatory, and defense responses Table .No GO terms that were statistically significant by FDR p-value were identified by GOrilla in males of the F1 2 mg/kg-day, F1 0.2 mg/kg-day, F2 0.033 mg/kg-day, or F3 0.033 mg/kg-day groups compared to oil controls.For F1 0.033 mg/kg-day exposed males compared to oil controls, GOrilla identified 45 GO terms, of which 40 were non-redundant and of which 14 had statistically significant FDR p-values Table . SignifiFor F2 2 mg/kg-day exposed males compared to oil controls, GOrilla identified 82 GO terms, of which 61 were non-redundant and of which 37 had statistically significant FDR p-values Table . For F2 p-values , female F3 0.03 mg/kg-day BaP (FDR P < 0.04), and female F3 0.2 mg/kg-day BaP (FDR P < 0.001) to respective F1 and F3 controls, and Tdgf1 was the top DEG when comparing female F3 2.0 mg/kg-day BaP to F3 vehicle controls (FDR P = 0.017). Consistent with these results, “Nodal signaling pathway” was the fourth most significant GO term in the female F2 2.0 mg/kg-day BaP females, and “positive regulation of activin receptor signaling pathway” was the tenth most significant GO term in the F1 0.2 mg/kg-day females, compared to respective controls.Among the male PGC genes with increased expression in female BaP-exposed E13.5 PGCs compared to vehicle controls were P = 0.045, effect of BaP dose by linear regression; Fig. P = 0.034, Mann Whitney test; Fig. P = 0.048, effect of BaP dose by linear regression; Fig. We performed immunofluorescence staining for TDGF1 and LEFTY2 and quantified expression using confocal microscopy and Imaris image analysis in F1 and F3 E13.5 vehicle and 0.033 and 2 mg/kg-day BaP ovaries. In the F1 ovaries, TDGF1 immunofluorescence increased with BaP dose in germ cell cytoplasm . gDNA of both male and female PGCs in E13.5 embryos was globally and strongly demethylated except for the IAP family of endogenous retroviruses, agreeing with previous studies29 Fig. A,B. NeitTo examine DNA methylation of the repetitive sequences in the mouse genome in more detail, we determined the genome-wide coverage of the RepeatMasker-registered repetitive sequences (GRCm38/mm10) by our WGBS. We identified the top 35 largest groups of repetitive sequences registered in the RepeatMasker file, 9 and 11 of which belonged to the LINE-1 family and B1-B4 repeats, respectively, whereas only one belonged to the IAP family (IAPEz-in indicated by asterisk) Fig. C. In conOur results demonstrate that in utero exposure via oral dosing of the PAH BaP to the pregnant F0 female mouse decreases the finite ovarian reserve in her directly exposed F1 daughters and in her unexposed F3 great grand-daughters. To the best of our knowledge, this is the first report of transgenerational effects on ovarian reserve by exposure to any PAH. Our E13.5 PGC RNA-seq results show that this is associated with statistically significant transgenerational upregulation in female PGCs of genes normally expressed highly in male PGCs and minimally expressed in female PGCs. In contrast, only modest effects were observed on sex-specific gene expression in male PGCs. BaP exposure did not affect the global DNA demethylation of gDNA that is expected in F1 E13.5 PGCs of both sexes. However, we observed increased methylation of B1-B4 repetitive sequences in BaP-exposed F1 and F3 offspring.1. The daily mutagenic PAH intake of a highly, non-occupationally exposed woman is about 10 µg/day from smoking 40 cigarettes, 3 µg/day from breathing urban air, and 17 µg/day from eating grilled/smoked foods; or a total of 0.5 µg/kg-day for a 60 kg woman, for a cumulative dose over 39 weeks of pregnancy of 137 µg/kg45. 0.033 mg/kg-day is about tenfold higher than the average daily dose of mutagenic PAHs of a highly exposed woman after allometric interspecies dose correction46. With our 10-day gestational dosing window, the cumulative dose of 0.33 mg/kg is about one third of the estimated cumulative dose during pregnancy of a highly exposed woman after allometric interspecies correction. Remarkably, this dose resulted in 50% fewer germ cells at E13.5 and primordial follicles at PND21 in the F3 female offspring. Prior studies have shown distribution of BaP and its metabolites to ovaries and embryos after oral and inhalational dosing. Oral dosing of pregnant female mice from E6.5–11.5 with two of the doses used in the present paper, 0.2 and 2 mg/kg-day, resulted in total BaP metabolite concentrations in the whole female E13.5 embryos of 1.9 and 2.6 ng/g tissue, respectively; due to tissue requirements, it was not possible to measure BaP metabolites in embryonic ovaries20. In contrast, metabolite concentrations in the placentas were about half those in the embryos20. The results were consistent with efficient transfer of BaP and its metabolites via the placenta and embryonic metabolism of BaP20. BaP metabolites were detectable in the ovaries of adult F344 rats as many as 28 days after a single 5 mg/kg oral dose, while the parent compound was detectable at 7 days, and was no longer detectable at 14 or more days after dosing47. In female F344 rats exposed to 3 doses of BaP via inhalation for 4 h/day for 14 days, BaP metabolite concentrations in ovaries in the highest dose group (100 μg/m3) were about ninefold higher than lung and about threefold higher than liver48.This is the first study to demonstrate multi- or trans-generational effects of a PAH at a human relevant dose, 0.033 mg/kg-day. BaP is a prototypical mutagenic PAH, which undergoes similar metabolism and has similar mechanisms of action as other mutagenic PAHs49. Therefore, unlike males, who have germline stem cells present in the testes throughout life, females are born with a finite number of germ cells, called oocytes, which are packaged into primordial follicles during the first postnatal week in mice49 and by the end of the second trimester in humans50. Depletion of the ovarian reserve of primordial follicles results in premature cessation of ovarian function. When this occurs in women prior to the age of 40, it is called premature ovarian insufficiency (POI), and it is a major cause of infertility and impaired fecundity in women53. POI is associated with increased risk of multiple adverse health outcomes. Women with POI have increased risk of dyslipidemia, endothelial dysfunction, thromboembolism, hypertension, metabolic syndrome, coronary artery disease, and stroke55. Women with POI also have increased risk of cognitive decline and Alzheimer’s disease58, and osteoporosis60. While it has been known for some time from experimental studies that preconceptional and gestational exposure to BaP and other PAHs depletes the ovarian reserve of primordial follicles and causes infertility in the F1 female offspring61, the data presented herein support that ancestral exposure to PAHs may be a heretofore unappreciated cause of POI. This is significant not just for fertility, but also for the overall health of women, given the adverse effects of POI on cardiovascular, brain, and bone health47.In mice and humans, germ cells in the developing ovaries enter meiosis and arrest in the diplotene stage of the first meiotic prophase prior to birthTdgf1 and Lefty2, which are components of the Nodal signaling pathway, were significantly upregulated in female PGCs across generations and BaP dose groups. Further supporting that BaP transgenerationally upregulates Nodal signaling in female germ cells, Nodal receptor genes, Acvr1b, and Acvr262, were also upregulated in several female BaP-exposed groups. Nodal signaling prevents male PGCs from entering meiosis during the developmental stage when female germ cells are beginning to enter meiosis64. GO analysis identified GO terms related to meiosis that were affected by F0 BaP exposure across generations and doses in females. Among the genes associated with the meiosis GO terms were Meiob, Syce1 and 3, Sycp2 and 3, Tex11, Tex12, Tex15, and Ccnb3, which were down-regulated in F1 2 mg/kg-day and F3 0.2 mg/kg-day females compared to respective controls. All of these, except Meiob and Ccnb3, are synaptonemal complex components65. Ccnb3 is required for the initiation of anaphase I in oocyte meiosis66. Meiob is a single strand DNA binding protein, which is required for meiotic recombination67. Taken together these results suggest that meiosis initiation in female germ cells directly or ancestrally BaP exposed may be disturbed and that this may play a role in the transgenerational ovarian phenotype. Future studies should undertake detailed analyses of meiosis I progression in embryonic ovaries directly or ancestrally exposed to BaP.The transgenerational depletion of the ovarian reserve caused by F0 BaP exposure was associated with statistically significant transgenerational increases in expression of male PGC marker genes in the female E13.5 PGC transcriptomes. Two male PGC marker genes, p-values < 0.05 between BaP-exposed and respective control groups revealed many GO terms related to immune responses and inflammation. Among the genes driving these GO terms were multiple c–c motif chemokines  and to a lesser extent their G protein-coupled receptors , which were increased across multiple BaP dose groups and generations. We previously reported increased expression of Ccl2 and Ccl7 by RNAseq of whole F1 E13.5 ovaries exposed to BaP from E6.5–11.5, confirmed with immunofluorescence for CCL220. CCL2, CCL3, CCL7, and CCL12 are monocyte chemoattractants68, which contribute to the tumor microenvironment of ovarian cancers71. Ccl2 expression was reportedly upregulated in cultured Type II alveolar pneumocytes by another PAH, 1-methylanthracene72. In the neonatal rat ovary inflammation induced by lipopolysaccharide administration caused ovarian reserve depletion73. Our results suggest that F0 BaP exposure may cause inflammation in the developing ovaries across generations.Analyses of DEGs that differed by at least 1.5-fold and had unadjusted 36. The F2 and F3 mice for Experiment 2 in the present paper were descended from the siblings of those F1 females. Interestingly, decreased ovarian reserve was not observed in the F2 and F3 females in this experiment. We think that the most likely reason is the shorter dosing windows and that exposure during both developmental windows is necessary for the transgenerational effect on ovarian reserve. The mice for Experiment 1 were transgenic OG2 mice on a C57BL/6J genetic background, while those for Experiment 2 were wild type C57BL/6J mice. However, we think it is unlikely that this genetic difference is the reason for the lack of transgenerational effects. Using mice heterozygous for the Glutamate Cysteine Ligase Modifier subunit, Gclm+/− mice, which are also on a C57BL/6J genetic background, we found that follicles per ovary were significantly decreased in F2 Gclm+/− mice descended from F0 mothers dosed for the longer, E6.5–15.5 dosing window with 2 mg/kg-day BaP  promoter and distal enhancer 2Mnn/J; referred to as OG2 mice35) were originally purchased from Jackson Labs (Strain #004654) and have been backcrossed 9 times onto a C57BL/6J genetic background and maintained as heterozygotes in our colony. OG2 mice were used for Experiment 1. Wild type C57BL/6J mice  were used for Experiment 2. Mice deficient in Gclm and littermate controls on a C57BL6/J background were used for Experiment 374. Mice were housed in an American Association for the Accreditation of Laboratory Animal Care International-accredited facility at the University of California Irvine (UC Irvine), with free access to deionized water and soy-free laboratory chow , on a 14:10 h light–dark cycle. Temperature was maintained at 21–23 °C. The experimental protocols were carried out in accordance with the Guide for the Care and Use of Laboratory Animals75 and ARRIVE Guidelines76 and were approved by the Institutional Animal Care and Use Committee at UC Irvine.Mice with germ cell-specific expression of EGFP under control of a Experiment 1, Transgenerational effects of in utero BaP on gonadal germ cell numbers and PGC transcriptomes and methylomes: Homozygous 10–14 weeks old male and female OG2 mice were mated on the evening of the female’s proestrus, determined by daily vaginal cytology. The next morning females with vaginal plugs were considered to be pregnant and that time point was designated embryonic day (E) 0.5. Pregnant F0 dams were fed sesame oil containing 0, 0.033, 0.2, or 2 mg/kg-day BaP  by oral pipetting into the cheek pouch daily from E6.5–11.5 for collection of PGCs or whole gonads from E13.5 F1 embryos or from E6.5–15.5 for generation of F2 and F3 offspring. We previously estimated that a highly environmentally exposed woman is exposed to 0.5 µg/kg-day of mutagenic PAHs, including BaP, for a cumulative dose of 1.0 mg over the course of a 39 week pregnancy, which is equivalent to 3.7 µg/kg-day in mice after allometric interspecies correction77. The 0.033 mg/kg-day dose in the present study provides a cumulative dose of 0.33 mg over 10 days, which is about one third of the estimated cumulative dose of mutagenic PAHs during pregnancy of a highly environmentally exposed woman. F1 and F2 non-littermate males and females from the same F0 dose group were mated to generate F2 and F3 generations, respectively. F1, F2, and F3 females were euthanized at PND21 for ovarian follicle enumeration. Pregnant F0, F1, and F2 dams were euthanized by CO2 asphyxiation on E13.5 for F1, F2, and F3 embryonic gonad collection and PGC isolation.Experiment 2, Transgenerational effects of BaP exposure during mitotic or meiotic ovarian developmental window: C57BL/6J male and female mice were mated on the evening of the female’s proestrus as described above, and pregnant females were orally dosed with 2 mg/kg-day BaP or oil vehicle from E6.5–11.5 (germ cell mitotic window during ovarian development) or from E12.5–17.5 (germ cell meiotic window of ovarian development). F0 dams were allowed to give birth to the F1 generation. To produce subsequent generations, at least one F1 female from each litter was mated with a non-littermate F1 male of the same treatment group to yield the F2 generation. F2 females were mated with non-littermate F2 males of the same treatment group to produce the F3 generation. F1 and F3 females were euthanized on the day of first vaginal estrus (puberty onset78), determined by vaginal cytology, and F2 females were euthanized at 4 to 5 months of age for ovarian follicle counts. Follicle count data from pubertal F1 females have already been reported 36.Experiment 3: Effects of F0 BaP exposure on ovarian follicle numbers in F2 offspring: Pregnant F0 female Gclm+/− mice, which had been mated with Gclm+/− male mice, were dosed with 0 or 2 mg/kg-day from E6.5–15.5, and F1 offspring were mated as for Experiment 1 to generate F2 female offspring. Gclm+/- F2 female offspring were euthanized at 4–5 months of age for ovarian follicle enumeration.79. E13.5 germ cells were identified based on their morphology  counting frames that were superimposed onto the sections in sampling grids subdivided into 15,625 μm2 (125 μm × 125 μm) squares. Germ cells were counted only if the germ cell fell within the counting frame and/or touched the inclusion boundaries and did not touch the exclusion boundaries. Lastly, the optical dissector height was set to 10 μm with guard zones on the top and the bottom of the section to account for irregularities of the sections. By multiplying the raw counts by the reciprocals of the counting fractions, the number of germ cells in the entire gonad was estimated. For PND 21 and pubertal ovaries, primordial, primary, and secondary follicle numbers were similarly counted. Three levels of sampling were used to determine the estimated number of follicles in the ovary- every 3rd section of the ovary with 10,000 μm2 (100 μm × 100 μm) counting frames and 30,625 μm2 (175 μm × 175 μm) sampling grids. Follicles were counted if the oocyte fell within the counting frame and/or touched the inclusion boundaries within the middle 10 μm of the sections. Antral follicles were followed through every section to avoid double counting using an Olympus BX-60 light microscope. Follicles were classified as primordial , primary , secondary (more than one layer of granulosa cells with no antrum), or antral (multiple layers of granulosa cells with antrum) follicles. Atretic follicles were identified as previously described81.Randomly selected E13.5 ovaries and testes from F1-F3 embryos and ovaries from post-natal females were harvested and immediately fixed in Bouin’s solution at 4 °C for 3 h, washed in 50% ethanol three times, and stored in 70% ethanol until embedding in glycolmethacrylate resin . Embedded E13.5 gonads and postnatal ovaries were sectioned at 20 µm and stained with hematoxylin and eosin. Stereological methods were used to obtain unbiased estimates of germ cell (E13.5) or ovarian follicle (PND21) numbersogy Fig. . Germ ce20, and the gonads were subjected to digestion using Embryoid Body Dissociation Kit  at 37 °C to obtain single cell suspensions. Enzymatic and physical dissociation was stopped by adding DMEM medium supplemented with 10% fetal calf serum. PGCs expressing EGFP from the germline-specific Pou5f1 promoter were enriched by FACS as we previously described39. Briefly, the resuspended cells were filtered through a 35 µm nylon mesh  and supplemented with propidium iodide (2 µg/ml) to remove dead cells. We first excluded cell debris and cell clumps , then gated out dead cells using propidium iodide  using the oligo(dT) primer provided in the kit, and Illumina sequencing libraries were generated using Low Input DNA Library Prep Kit (TAKARA Bio). Libraries were sequenced using an Illumina NextSeq 500 deep sequencer with high output flowcells to obtain 75 + 75 nt paired-end FASTQ reads, which were subjected to quality control analysis using the fastQC tool . After adaptor sequences and low-quality reads (< 30) were removed using the Trim Galore! tool, FASTQ reads were aligned to the GRCm38/mm10 mouse reference genome using the STAR aligner software83. The mRNA expression counts were normalized using the negative binominal trimmed mean of M-values (TMM) method implemented by the Bioconductor package edgeR84. The normalized counts were subjected to unsupervised hierarchical clustering analysis and visualization using Cluster85 and Java TreeView86. Differentially expressed genes (DEGs) were identified using the generalized linear model likelihood ratio test implemented by edgeR84. For determination of sex-specific DEGs, male and female PGCs isolated from unexposed with a set of criterion of false discovery rate (FDR) < 0.05 and greater than twofold changes. Exposure-specific DEGs were sorted by statistical significance (most significant genes were ranked at the top).To evaluate expression of mRNA transcripts, aligned reads in the BAM format were assigned to exons of the mm10 gene model and counted using the Bioconductor package Rsubread87 and confirmed > 99%. Libraries were sequenced using an Illumina NovaSeq 6000 deep sequencer with S4 flowcells to obtain 150 + 150 nt paired-end FASTQ reads, which were subjected to quality control analysis using fastQC. After adaptor sequences and low-quality reads (< 30) were removed using the Trim Galore!, FASTQ reads were analyzed using the Bismark pipeline as we previously described to obtain genome-wide profiles of CpG methylation88. Approximately 100 million uniquely mapped leads were obtained for each library. RepeatMasker-estimated coordinates of repetitive sequences in the GRCm38/mm10 mouse reference genome were downloaded from the UCSC browser, and methylation profiles of each repetitive sequences were determined by R scripts.gDNA was isolated from the FACS-enriched F1 and F3 PGCs using AllPrep Micro Kit and subjected to synthesis of bisulfite-converted Illumina sequencing libraries using EZ DNA Methylation-Gold kit  and Accel-NGS® Methyl-Seq DNA Library Kit . Bisulfite conversion efficiency was evaluated as we previously describedE13.5 gonads were fixed in 4% PFA for 2–3 h at 4 °C and cryoprotected in 30% sucrose prior to embedding in OCT and freezing. The frozen gonads were cryosectioned at 7 μm thickness. Sections were pretreated in 10 mM citric acid for 15 min at 95 °C, blocked in PBS-Triton X-100 solution with 5% normal goat serum and 1% BSA for 1 h and incubated with primary antibodies, rabbit anti-TDGF1 , rabbit anti-LEFTY2 , and rat anti-TRA98 , at 4 °C overnight. After washing, the primary antibodies were detected with corresponding secondary antibodies, goat anti-rabbit Alexa Fluor 633  and goat anti-rat Alexa Fluor 488 , for 1 h at room temperature. Sections were treated with autofluorescence quenching reagent  and stained with DAPI for nuclei staining. Slides were mounted in Prolong glass antifade mountant . Images of fluorescently TRA98-labeled germ cells with TDGF1 or LEFTY2 were generated using Zeiss LSM 900 Airyscan 2 microscope and processed using the Bitplane Imaris imaging software (v.9.9.0). Image J was used to quantify mean fluorescence intensity of TDGF1 or LEFTY2 immunopositive cells in a whole ovary section by an investigator blind to experimental group. Briefly, images were converted to 16-bit grayscale and the whole ovary was selected using “Polygon” tool. Then, the mean intensity of fluorescence signal was measured and used for data analysis.P < 0.05. Analyses were carried out using SPSS 28 for Mac or R.Data are presented as mean ± SEM unless otherwise noted. For germ cell and ovarian follicle counts, Generalized Estimating Equations with exchangeable correlation matrices were used to adjust for correlation among counts in females from the same litter or, for F2 and F3 females, descended from the same F0 dam. For immunofluorescence quantitation, linear regression and Kruskal Wallis test were used to examine the effects of BaP dose. For comparisons of two means, t-tests or non-parametric Mann Whitney tests were used, as appropriate. Statistical significance was set to Permutation tests were performed to evaluate the likelihood of randomly selecting sex-specific DEGs Table  out of aSupplementary Figures.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.Supplementary Table S7.Supplementary Table S8.Supplementary Table S9.Supplementary Table S10.Supplementary Table S11.