Archives of Toxicology

Few protocols are available for retrieving male germ cell-specific information on DNA damage dynamics or assessing the potential harmful effects of environmental contaminants, drugs, or lifestyle factors related to genotoxicity. Here, we present a protocol for evaluating testicular germ cell genotoxicity using a modified version of the alkaline comet assay in rodent testicular germ cells. The protocol includes experimental design, preparation of testicular cell suspensions, comet analysis, germ cell-specific scoring, and data curation methods to collect information on testicular male germ cells, specifically spermatids and primary spermatocytes. For complete details on the use of this protocol please refer to Olsen et al., in preparation HighlightsO_LIDNA damage levels can be specifically measured in testicular germ cells using the developed revised version of the alkaline comet assay C_LIO_LI1C spermatids as well as 4C primary spermatocytes can be assessed C_LIO_LIBoth manual- and modeling-based approaches were developed that facilitate user-friendly protocols to select 1C spermatid comets. C_LIO_LIThis protocol expands the limited methodologies available to study germ cell DNA damage dynamics C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/624648v7_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1c18b1eorg.highwire.dtl.DTLVardef@1949f12org.highwire.dtl.DTLVardef@576e1eorg.highwire.dtl.DTLVardef@1feb14b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

Toxicity testing is under transformation as it aims to harness the potential of New Approach Methods (NAMs) as alternative test methods that may be less resource intensive (i.e., fewer animals, cheaper costs, quicker assays) than traditional approaches while also providing more data and information. While many stakeholders are of the opinion that this unfolding transformation holds significant promise as a more efficient and ethical way forward, few studies have compared the resources required for NAMs versus those needed for traditional animal-based toxicity tests, particularly in the field of ecotoxicology. The objective was to compare resources needed for traditional animal-based ecotoxicity tests versus alternative tests using emergent NAMs. From a bibliometric review, we estimate that traditional tests for a single chemical cost $118,000 USD, require 135 animals, and take 8 weeks. In comparison, alternative tests cost $2,600, require 20 animals (or none), and take up to 4 weeks to test 16 (to potentially hundreds of) chemicals. Based on our analysis we conclude that NAMs in ecotoxicology can be more advantageous than traditional methods in terms of resources required (i.e., monetary costs, number of animals needed, and testing times). We note, however, that the evidence underpinning these conclusions is relatively sparse. Moving forward, groups developing and applying NAMs should provide more detailed accounts of the resources required. In addition, there is also a need for carefully designed case studies that demonstrate the domain of applicability of NAMs (and make comparisons to traditional tests) to ultimately build confidence among the user community.

Biochemical investigations of the hepatoprocarcinogen N-acetyl-2-aminofluorene (AAF) have shown that normal adult rat hepatocytes in primary culture express two sets of pharmacokinetic constants - designated Systems I and II, and Sites I and II - associated respectively with the metabolism (System I [high-affinity Km[APPARENT] and low-velocity VMAX[APPARENT]] and System II [low-affinity Km APPARENT] and high-velocity VMAX[APPARENT]]), and the macromolecular binding (Site I [high-affinity KD[APPARENT] and low capacity BMAX[APPARENT]] and Site II ([low-affinity KD[APPARENT] and high-capacity BMAX[APPARENT]]) of AAF. Additional findings - that genomically saturating levels of AAF-DNA adducts form far below reported extracellular AAF concentrations required to block replicative and repair DNA synthesis; and, that biphasic Site I and Site II BMAX[APPARENT] and KD[APPARENT] expression curves varied inversely with respect to time and magnitude during hepatocyte growth - led us to wonder how macromolecular binding constants are expressed during chemical hepatocarcinogenesis. These questions were addressed by Scatchard analysis measurements through five consecutive AAF feeding cycles. Notably, cultured premalignant hepatocytes displayed reduced and elevated BMAX[APPARENT] and KD[APPARENT] levels, respectively, akin to the Site I and Site II expression curves observed during hepatocyte growth transitions in vitro. In contrast, prominent hepatocellular functions such as N-OH-AAF production, DNA replication, cell aggregation and resistance to AAF toxicity displayed different temporal trajectories. Impact StatementStriking similarities are observed between both Site I and Site II BMAX and KD expression curves during in vitro and in vivo premalignant growth transitions. These new findings fit earlier ones that hepatocytes growing during carcinogen exposure manifest fewer intranuclear AAF-DNA adducts. How this phenomenon leads to malignancy remains unclear.

Obesity and dietary fat consumption are associated with increased risk for certain cancers. We conducted a set of experiments designed to investigate both the inherent mutagenicity of high-fat diet (HFD) and the effect of HFD exposure on sensitivity to ethyl carbamate, a known mutagen. Greater body mass in females was associated with higher frequency of Pig-a mutant erythrocytes and HFD-induced obese female mice exhibited increased sensitivity to ethyl carbamate-induced mutagenesis compared to control mice. Together, these findings indicate that HFD exposure induces both endogenously generated changes related to mutagenesis and interacts with the mutagenic effects of ethyl carbamate.

The clinical use of the anticancer drug doxorubicin (Dox) is limited by irreversible cardiotoxicity. The detailed molecular mechanisms involved and the pathophysiological relevance of different cardiac cell types, including progenitor cells, are still unclear. Here, we investigated stress responses of murine embryonic stem cells (mESC), endothelial progenitor cells (EC d4) and terminally differentiated endothelial-like cells (EC d6) following exposure to Dox and selected pharmacological inhibitors of DNA repair and DNA damage response (DDR) (RAD51i B02 and HDACi entinostat (EST)). We found that EC d4 exhibited a pronounced Dox hypersensitivity as compared to both mESC and EC d6, which was independent of drug transport. Analysis of EdU incorporation and replication fork progression following drug treatment revealed substantial agent-specific differences between mESC and the various differentiation stages. Furthermore, cellular susceptibility to drug-induced formation of DNA damage (i.e. DSB and SSB) also changes with ongoing differentiation and drug treatment, with mESC and EC d4 / EC d6 being particular prone to enhanced residual SSB and DSB levels, respectively. Dox treatment of EC d4 did not affect their differentiation into EC d6, but caused multiple functional impairments of the surviving EC d6 progeny, including defects in mitochondrial homeostasis, barrier function related to cell-cell adhesion factors ZO1 and VE-cadherin, response to cytokine stimulation as well as LDL uptake. To summarize, we show substantial differences in the response of mESC, EC d4 and EC d6 to Dox and pharmacological inhibitors of DNA repair and DDR. Most important, treatment of EC d4 results in pronounced persisting functional impairments of differentiated EC d6, pointing to a transient particularly drug-sensitive time window during endothelial differentiation. These findings are important for hazard assessment in developmental toxicology and regenerative medicine in the context of anticancer drug-induced normal tissue damage. Highlights- EC d4 are most vulnerable towards Dox-induced cytotoxicity independent of drug transport - EC d4 and EC d6 display higher steady-state levels of drug-induced DSB as compared to mESC - Drug-induced replication stress is highest in mESC and decreases with differentiation. - Both Dox and DNA repair/DDR inhibitors damage mitochondrial homeostasis in EC d4 and EC d6 - Terminally differentiated EC d6 derived from drug-treated EC d4 display multiple functional impairments

N-Nitrosodimethylamine (NDMA) is classified as an animal and probable human carcinogen. Murine liver DNA adducts, mutations, MGMT and CYP2E1 were evaluated following chronic administration of NDMA in drinking water. In a dose-escalation study, 7-methylguanine (m7G) increased linearly with NDMA dose. O6-Methylguanine (m6G) remained near background for NDMA doses up to [~]1 ppm, beyond which its level, and corresponding mutations, rose steeply. An extended study was done with 5 ppm NDMA, in which adducts were measured at 3 weeks and mutations at 10 weeks. While the level of CYP2E1 was unchanged, MGMT gene transcription was induced in females at 10 weeks. Homologous recombination-mediated chromosomal rearrangements did not increase over background. Point mutations, however, were elevated substantially in both sexes. Mutational analysis over 96 trinucleotide contexts revealed predominantly GC[->]AT mutations in 5-purine-G-3 contexts in a pattern matching human COSMIC cancer mutational signature SBS11, with secondary features resembling SBS119 (AT[->]GC). Taken together, the data implicate m6G as the dominant mutagenic adduct under chronic dosing with NDMA. Furthermore, the genomic m6G level was identified at which its dedicated repair protein, MGMT, became saturated. The coordinated application of DNA adduct, mutational and biochemical analyses provides a new approach for early detection and cancer management. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/687536v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1e6ae72org.highwire.dtl.DTLVardef@1c6fad6org.highwire.dtl.DTLVardef@7e3716org.highwire.dtl.DTLVardef@52a87b_HPS_FORMAT_FIGEXP M_FIG C_FIG

In regulatory toxicology an outcome is claimed positive when both a trend is significant and any pairwise test against control. Two statistical approaches are proposed: a joint Dunnett and Williams test (assuming the dose as a qualitative factor) and a joint test of the Tukey regression test and Dunnett test (assuming the dose as a quantitative covariate). Related R software is available.

An important element of the European Unions "Registration, Evaluation, Authorisation and Restriction of Chemicals" (REACH) regulation is the evaluation by the European Chemicals Agency (ECHA) of testing proposals submitted by the registrants to address data gaps in standard REACH information requirements. The registrants may propose adaptations, and ECHA evaluates the reasoning and issues a written decision. Read-across is a common adaptation type, yet it is widely assumed that ECHA often does not agree that the justifications are adequate to waive standard testing requirements. From 2008 to August 2023, a total of 2,630 Testing Proposals were submitted to ECHA; of these, 1,538 had published decisions that were systematically evaluated in this study. Each document was manually reviewed, and information extracted for further analyses. Read-across hypotheses were standardized into 17 assessment elements (AEs); each submission was classified as to the AEs relied upon by the registrants and by ECHA. Data was analyzed for patterns and associations. Testing Proposal Evaluations (TPEs) with adaptations comprised 23% (353) of the total; analogue (168) or group (136) read-across adaptations were most common. Of 304 read-across-containing TPEs, 49% were accepted; the odds of acceptance were significantly greater for group read-across submissions. The data was analyzed by Annex (i.e., tonnage), test guideline study, read-across hypothesis AEs, as well as target and source substance types and their structural similarity. While most ECHA decisions with both positive and negative decisions on whether the proposed read-across was adequate were context-specific, a number of significant associations were identified that influence the odds of acceptance. Overall, this analysis provides an unbiased overview of 15 years of experience with testing proposal-specific read-across adaptations by both registrants and ECHA. These data will inform future submissions as they identify most critical AEs to increase the odds of read-across acceptance.

Benzo[a]pyrene diol epoxide (BPDE) is a metabolite of the environmental contaminant Benzo[a]pyrene- a byproduct of incomplete combustion of organic matter. BPDE reacts with DNA to form BPDE-DNA bulky adducts which if not removed can lead to mutations due to DNA base-pair substitutions. While the effects of BPDE on somatic cells are fairly well described, its effects on early human development are currently unknown. In this study, we investigated for the first time the effect of BPDE on human induced pluripotent stem cells (hiPSCs) and their differentiated neuroprogenitor cells (NPCs) as a model for early embryonic development. Furthermore, we compared hiPSCs and NPCs derived from cells of patients suffering from Nijmegen Breakage Syndrome (NBS), which is a chromosomal instability disorder characterized by defective DNA repair and increased risk of malignancies. Transcriptome analysis, coupled with protein content analysis employing immunostaining and Western blots, revealed that hiPSCs are more sensitive to BPDE exposure when compared to NPCs with an enhanced expression of several genes associated with p53-mediated DNA damage response, including DNA repair by lesion bypass, cell cycle checkpoints and extrinsic apoptosis. We also identified that cells from NBS patients showed less apoptotic response and a distinct p53 response than their healthy counterparts. This iPSC-based study enhances our meagre knowledge of the effects of BPDE on early human development in both healthy individuals and NBS patients. Furthermore, our model conforms with the 3Rs principle.

The transition toward animal-free safety assessment of chemicals has accelerated the development of New Approach Methodologies (NAMs) for predicting skin sensitization. However, individual in silico models and experimental NAM assays frequently produce inconsistent or contradictory results, limiting their reliability when used in isolation. To address this challenge, we present a tiered integrated assessment framework implemented through the open source SaferSkin application, which enables systematic comparison and integration of multiple predictive models and experimental data within a transparent weight-of-evidence workflow. In this case study, a diverse set of 21 reference compounds was evaluated using a battery of in silico approaches, including the OECD QSAR Toolbox, VEGA, CASE Ultra and additional machine-learning models implemented within SaferSkin. The platform enables side-by-side comparison of predictions and integration of experimental data through Bayesian network models, allowing probabilistic updating of predictions as new evidence becomes available. Our results demonstrate that reliance on any single predictive model is insufficient for robust hazard identification due to frequent disagreement between models. In contrast, consensus interpretation across multiple modelling approaches combined with targeted experimental evidence substantially improves predictive confidence. The integrated weight-of-evidence framework showed strong concordance with reference classifications and was further supported by independent validation using the Pred-Skin Bayesian model. Importantly, the tiered workflow enables resolution of ambiguous cases. For example, lower-tier predictions for ethyl (2E,4Z)-deca-dienoate were inconsistent across models, whereas targeted third-tier testing using the SENS-IS assay identified the compound as a strong sensitiser (GHS Category 1A). Overall, this study demonstrates how integrated modelling, Bayesian evidence updating and targeted NAM testing can reduce uncertainty in skin sensitization assessment. The SaferSkin framework provides a transparent and reproducible approach for implementing Next Generation Risk Assessment (NGRA) strategies and supports the development of animal-free regulatory toxicology and Safe-and-Sustainable-by-Design chemical innovation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/711911v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@b59ca0org.highwire.dtl.DTLVardef@13de455org.highwire.dtl.DTLVardef@599358org.highwire.dtl.DTLVardef@d87fd1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

Nicotinamide riboside chloride (NR-Cl) has been studied predominantly by the oral route, while information regarding its toxicity following parenteral administration is limited. To characterize route-dependent acute toxicity and estimate median lethal doses (LD50), pharmaceutical-grade NR-Cl was evaluated following bolus intravenous (IV), intramuscular (IM), and subcutaneous (SC) administration in female Sprague Dawley rats, in three independent studies. All studies were conducted using an adapted OECD Guideline 425 Up-and-Down procedure, modified for parenteral administration, in the absence of standardized route-specific OECD guidance. Animals received a single dose of NR-Cl via the respective administration route and were monitored for mortality, clinical signs, bodyweight changes, and gross pathological findings over a 14-day observation period. Following IM and SC administration, no mortalities were observed at doses up to 2000 mg/kg, and LD50 values for both routes were determined to be greater than 2000 mg/kg. In contrast, IV administration yielded an estimated LD50 of approximately 2000 mg/kg. These findings demonstrate that the acute toxicity of NR-Cl differs by route of administration and establish foundational safety benchmarks to support future research.

As excretion organs, the kidneys and liver are exposed to high concentrations of potentially toxic substances. While animal models remain the gold standard for organ-specific toxicity testing, alternative ex vivo approaches are essential to align with the 3R principles (refinement, reduction, replacement). Precision-cut tissue slices (PCTS) retain native tissue architecture, cellular heterogeneity, the interplay of different cell types, and metabolic capacity, offering a promising link between in vitro and in vivo models. Here, we aimed to establish an optimized protocol for preparing and culturing precision-cut kidney and liver slices (PCKS and PCLS) from mice for use in substance-oriented toxicological tests. Key parameters - including slice thickness, media composition, oxygenation, glucose levels, and incubation time - were refined to maintain tissue viability and metabolic function. Five known toxins - acetaminophen, cyclosporin A, cisplatin, arsenic trioxide, and aristolochic acid I - were tested. While PCKS showed comparable sensitivity to established kidney cell lines, PCLS achieved IC50 values closely matching in vivo toxicity data. High reproducibility across different experimenters was achieved, highlighting the robustness of the model. In conclusion, this ex vivo system provides a valuable, reproducible, and ethically approved platform for acute nephrotoxicity and hepatotoxicity testing, supporting preclinical drug screening and potentially reducing reliance on animal experiments.

People are exposed to high concentrations of antibacterial agent cetylpyridinium chloride (CPC) via personal care and food products, despite little information regarding CPC effects on eukaryotes. CPC is used as an antibacterial agent via a detergent mechanism when above [~]600- 900 M. While three previous studies suggested CPC mitochondrial toxicity, this phenomenon is not well-studied. Here, we show that low-micromolar CPC inhibits mitochondrial ATP production in primary human keratinocytes, mouse NIH-3T3 fibroblasts, and rat RBL-2H3 immune mast cells, in galactose media, which causes cells to produce ATP via mitochondria. ATP inhibition via CPC (EC50 1.7LJ{micro}M) is nearly as potent as that caused by canonical mitotoxicant CCCP (EC50 1.2LJ{micro}M). CPC inhibition of oxygen consumption rate (OCR) tracks with that of ATP: OCR is halved due to 1.75 M CPC in RBL-2H3 cells and 1.25 M in primary human keratinocytes. Here we demonstrate that CPC is more potent as a mitotoxicant than as an immune mast cell signaling inhibitor, an effect published previously. Mitochondrial [Ca2+] changes can cause mitochondrial dysfunction. Here we show, using a novel plate reader assay with reporter CEPIA2mt, that CPC causes mitochondrial Ca2+ efflux from mast cells via an ATP-inhibition mechanism. Using super-resolution microscopy (fluorescence photoactivation localization) in live cells, we have discovered that CPC causes mitochondrial nanostructural defects in fibroblasts, including the formation of spherical structures with donut-like cross section, as quantified by novel Fourier transform analysis. This work reveals CPC as a mitotoxicant despite widespread use, highlighting the importance of further research into its toxicological safety.

Pluripotent stem cells (PSCs) possess unique characteristics that distinguish them from other cell types. Human embryonic stem (ES) cells are recently gaining attention as a powerful tool for human toxicity assessment without the use of experimental animals, and an embryonic stem cell test (EST) was introduced for this purpose. However, human PSCs have not been thoroughly investigated in terms of drug resistance or compared with other cell types or cell states, such as naive state, to date. Aiming to close this gap in research knowledge, we assessed and compared several human PSC lines for their resistance to drug exposure. Firstly, we report that RIKEN-2A human induced pluripotent stem (iPS) cells possessed approximately the same sensitivity to selected drugs as KhES-3 human ES cells. Secondly, both ES and iPS cells were several times less resistant to drug exposure than other non-pluripotent cell types. Finally, we showed that iPS cells subjected to naive-state induction procedures exhibited a sharp increase in drug sensitivity. Upon passage of these naive-like cells in non-naive PSC culture medium, their sensitivity to drug exposure decreased. We thus revealed differences in sensitivity to drug exposure among different types or states of PSCs and, importantly, indicated that naive-state induction could increase this sensitivity.

Ethylene oxide (EtO) is primarily used as an intermediate in the manufacture of chemicals, with a minor use as a sterilant for medical equipment and food products. It is a direct-acting alkylating agent that reacts with cellular macromolecules, including proteins and DNA. EtO has been shown to induce tumors in rodents and humans. DNA reactivity has been the postulated mode of action (MOA) for its carcinogenicity. The current study has investigated the dose response for EtO-induced genetic damage to inform the biological plausibility of a dose-response model for cancer risk assessment. Male and female B6C3F1 mice were exposed to 0, 0.05, 0.1, 0.5, 1, 50, 100, or 200 ppm EtO by whole-body inhalation (6 hours/day for 28 days, 7 days/week). Mutagenicity was assessed by determining the frequency of mutant Pig-a phenotype in reticulocytes (RET) and mature red blood cells (RBC) on Day 28. Cytogenetic damage was evaluated by the erythrocyte micronucleus (MN) test in blood samples collected on Days 5 and 28. EtO is a relatively weak genotoxicant with treatment-related increases in Pig-a and MN frequencies being seen primarily at 200 ppm. The hockey-stick shaped dose response for genetic damage may be conservatively interpreted as being no more than a linear response with a single slope. Thus, a cancer risk assessment dose-response model consisting of a single linear slope throughout the exposure range is biologically plausible and consistent if EtO were acting through a mutagenic MoA for its carcinogenicity.

Inter-individual and population variability in susceptibility to chemical exposures confounds determination of threshold exposure levels to protect the most vulnerable. Current risk assessment frameworks, in the absence of empiric chemical-specific data, generally recommend default or probabilistic adjustment factors to account for such variability. We present an experimental approach to incorporate common genetic variants potentially impacting population-level differences in toxicant susceptibility into human cell-based models for any cellular apical endpoint of interest. We focus on the genes with the most common aggregate loss-of-function (LoF) alleles in the gnomAD v3.0 data which we designated as the PopVarLoF set. Unexpectedly, enrichment analysis of these genes found significant overrepresentation of gene products playing important functional roles in toxicology. Interrogation of GWAS and PheWAS databases found that these genes are associated with diverse metabolic phenotypes consistent with the relevance of the PopVarLoF set in studying variability of toxicant response in human populations. We further characterized the PopVarLoF set by developing custom lentiviral CRISPR knockout libraries targeting the PopVarLoF genes to assess their functional essentiality in the HepG2/C3A cell line. Functional disruption of 14 of the PopVarLoF genes ([~]1 %) without toxicant exposure resulted in significant growth defects in this cell line, consistent with the majority of PopVarLoF gene products having non-essential roles. The development of human cell-based toxicity assays or other NAMs which include the empiric assessment of common genetic sources of population variability in susceptibility to chemical exposure could contribute to more robust risk assessment which protects vulnerable populations while reducing uncertainty. Impact statementWe characterize common loss of function genetic variants which could impact toxicant susceptibility and describe an approach to incorporate them into NAMs to enable empiric estimates of the contribution of genetic variability to diverse toxicity endpoints.

The zebrafish has been widely used as a predictive model in safety and toxicology. Low cost high-throughput screening can be achieved with this model, and the genome contains orthologues of the majority of human disease genes. However, previous studies indicate that the predictivity of the zebrafish model in toxicology varies between compound and compound class. We examined this issue by screening 24 compounds from two different compound classes, metals and biocides (pesticides/insecticides) for toxicity in the zebrafish model and looked at the effects on hatching, morphology and predictivity for mammalian toxicity. Wild-type zebrafish embryos were exposed to test compounds in 96-well plates for 96 hours starting at 24 hours post fertilization. Hatching was either delayed or accelerated depending on the compound. Three types of alteration in behavioural responses were noted: (i) hypoactivity; (ii) hyperactivity; and (iii) biphasic response (a dose-dependent shift between hypo- and hyperactivity). LC50 of compounds was calculated and compared to published LD50 values in rodents. The zebrafish-rodent values were poorly correlated for both metals and biocides. We conclude that, although the zebrafish is a good model for some aspects of toxicology, its predictivity for mammalian toxicity needs to be determined per compound class.

Evaluation of unintended immunotoxicity represents an important component of nonclinical safety assessment, as perturbation of immune function may increase susceptibility to infection, impair vaccine responses, and disrupt immune homeostasis. Regulatory guidance, including the ICH S8 Immunotoxicity Guideline, recommends a weight-of-evidence approach in which observations from conventional toxicological endpoints are integrated with functional immune assays to support interpretation of immune system effects. The present study applied an integrated immunotoxicity evaluation framework to examine concordance among structural, functional, and cellular immune endpoints in male Sprague-Dawley rats using a well-characterized immunosuppressive reference compound. Hematological evaluation revealed leukopenia characterized primarily by lymphocyte depletion. Reductions in spleen and thymus weights were accompanied by histopathological evidence of lymphoid depletion in multiple immune tissues, including spleen, thymus, lymph nodes, Peyers patches, and bone marrow. Functional immune competence was assessed through hemagglutination antibody response to sheep red blood cells and delayed-type hypersensitivity assays, both of which demonstrated marked suppression of adaptive immune responses. Flow cytometric immunophenotyping further demonstrated substantial reductions in B-cell populations and decreases in CD4 and CD8 T-cell counts, whereas NK cell populations were comparatively less affected. The concordance of hematological alterations, lymphoid tissue changes, impaired functional immune responses, and lymphocyte subset depletion provides integrated evidence of immune system perturbation. These findings demonstrate that complementary immunotoxicity endpoints collectively support hazard characterization of immune system effects under GLP conditions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/713556v1_ufig1.gif" ALT="Figure 1"> View larger version (72K): org.highwire.dtl.DTLVardef@beaf9dorg.highwire.dtl.DTLVardef@fb9f10org.highwire.dtl.DTLVardef@187ff06org.highwire.dtl.DTLVardef@1780dc2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Single doses of perfluoro-n-decanoic acid (PFDA) cause wasting, a progressive loss of 30 to 50% body weight, increasing liver/body weight ratios, and death within several weeks (Olson and Andersen, 1983). Repeat high doses of perfluorooctane sulfonate (PFOS) produce a subset of these responses in rats and monkeys. The mode of action (MOA) of these wasting-like syndromes is not clear, nor is it understood if these responses are limited to a subset of perfluoroacid substances (PFAS) or a common response to high dose exposure with a larger number of PFAS. To identify pathway perturbations in liver caused by PFAS, we analyzed published in vitro gene expression studies from human primary liver spheroids treated with various PFAS for treatment times up to 14 days (Rowan-Carroll et al., 2021). With treatment times of 10 to 14 days, longer-chain PFAS compounds, specifically PFOS, perfluorodecane sulfonate (PFDS) and higher doses of perfluorooctanoic acid (PFOA), downregulated large numbers of genes in pathways for steroid metabolism, fatty acid metabolism and biological oxidations. Shorter chain PFAS compounds upregulated genes in pathways for fatty acid metabolism. Although PFDA was more toxic and could only be examined at 1-day of treatment, it also downregulated genes for lipid metabolism, steroid metabolism, and biological oxidations. Shorter chain PFAS, both carboxylic and sulfonic acids, did not lead to downregulation of pathways for fatty acid or steroid metabolism. TCDD is also known to cause wasting responses in rodents and humans. In intact rats, high dose responses of longer chain PFAS produce downregulation of batteries of genes associated with fatty acid oxidation and lipogenesis similar to those seen with TCDD. Based on our results, when combined with other literature, we propose that the longer-chain PFAS impair lipogenic pathways through inhibitory interactions between PPAR{beta}, PPAR and PPAR{gamma}.

AO_SCPLOWBSTRACTC_SCPLOWPeripubertal exposure to the phthalate metabolite mono-(2-ethylhexyl) phthalate (MEHP) in rodents causes testicular inflammation, spermatocyte apoptosis, and disruption of the blood-testis barrier. The MEHP-induced inflammation response includes an infiltration of macrophages and neutrophils to the testes, although the cause and purpose of this response is unknown. Recently, a population of testicular macrophages phenotypically distinct from those resident in the interstitium was described in mice. Testicular peritubular macrophages aggregate near the spermatogonial stem cell niche and are believed to stimulate their differentiation. We hypothesized that if testicular peritubular macrophages do indeed stimulate spermatogonial differentiation, MEHP exposure would result in an increase of peritubular macrophages to stimulate the replacement of lost spermatocytes. Male rats were exposed to 700 mg/kg MEHP or corn oil (vehicle control) via oral gavage at PND 28 and euthanized at 48 hours, 1 week, or 2 weeks later. Tubules were stained with immunofluorescent markers for macrophages and undifferentiated spermatogonia. Peritubular macrophages were observed in rat testis similar to those previously described in mice: MHC-II+ cells on the surface of seminiferous tubules with heterogeneous morphology. Quantification of MHC-II+ cells revealed that, unlike in the mouse, their numbers did not increase through puberty. MEHP increased macrophage presence by six-fold 48-hours after exposure and remained elevated by two-fold two weeks after exposure. An increase of differentiating spermatogonia occurred two weeks after MEHP exposure. Taken together, our results suggest that peritubular macrophages play a crucial role in the testis response to acute injury and the subsequent recovery of spermatogenesis.\n\nSummary SentencePhthalate-induced testicular injury results in an increase of specialized peritubular macrophages that may assist in the recovery of spermatogenesis.