Abstract Cardiovascular (CV) disease is the leading cause of maternal mortality worldwide and has been partially linked to the cardiometabolic remodeling required to support fetal growth. In healthy pregnancies, these adaptations reverse postpartum without long-term consequences; however, impaired reverse remodeling (RR) increases future CV risk. Pregnant women living with HIV face additional risk, though it remains unclear whether this is driven by chronic low-level viremia or continuous antiretroviral therapy (ART). ART used during pregnancy often includes nucleoside reverse transcriptase inhibitors (NRTIs), which can affect mitochondria - organelles essential for maintaining cardiac function. Here, we tested the hypothesis that NRTI-containing ART negatively impacts the maternal heart. Using a rat model, we show that ART impairs pregnancy-associated cardiac RR. Mechanistically, ART induced mitochondrial alterations in both the heart and the liver, leading to dyslipidemia that collectively compromised cardiac function. These findings emphasize the importance of maternal cardiovascular health research and the need for further studies on the long-term effects of ART.

2,2'-Dimorpholinodiethyl ether (DMDEE) is a tertiary amine catalyst used in the production of polyurethane foams. Human exposure may occur occupationally through inhalation of DMDEE vapors. B6C3F1/N mice (5-6 weeks old) were exposed to 0-1000 mg DMDEE/kg in water by oral gavage daily for 4 weeks. Time-mated Harlan Sprague Dawley (HSD) rats (12-14 weeks old) were exposed to 0-500 mg DMDEE/kg in water from gestation day (GD) 6 through post-natal day (PND) 27 by oral gavage; their pups were directly dosed from PND12 through PND27. Blood was collected from mice (4-weeks), rat dams (GD18, PND4, PND28) and rat pups (PND 4, PND 28) for hematology, clinical chemistry, and micronucleus evaluation; mouse tissues were analyzed for histopathology. DMDEE (500-6000 μg/plate) was evaluated for mutagenicity using Salmonella typhimurium (TA98, TA100) or Escherichia coli WP2 uvrA pKM101. No DMDEE-related effects were observed on pregnancy or litter parameters in HSD rats. The mouse and rat micronucleus tests and the bacterial reverse mutation tests were negative. At 1000 mg DMDEE/kg, behavioral observations of circling, repetitive head lifting, and abnormal gait were accompanied by cytoplasmic vacuolization in epithelial cells of the brain choroid plexus, and in the renal cortical tubules in male and female mice. Cytoplasmic vacuolization was also observed in the epithelial cells in the coagulating gland, prostate, and epididymis of male mice. These data indicate deficits in the cellular structure and function of neurological and renal tissues in male and female mice, as well as male reproductive tissues at higher doses and suggest caution in the industrial use of DMDEE.

IntroductionGestational antiretroviral therapy (ART) has significantly reduced the risk of vertical transmission of HIV, but concerns linger about its long-term effects on the fetal immune system and intestinal health. Our previous work has demonstrated dose-dependent changes in the fecal and mucosa-associated microbiome of adult rat offspring perinatally exposed to TC-ART (tri-combination ART: dolutegravir, abacavir, and lamivudine). These changes may either be driven by alterations in immune system and intestinal barrier integrity or potentially impact them.MethodsIn this study, we further investigated the long-term effects of perinatal TC-ART exposure on intestinal permeability, cytokine profiles, and intestinal mucosa morphology.ResultsWe observed statistically significant sex-dependent differences, with male offspring exhibiting reduced weight gain, a dichotomous response between low and high dose for inflammatory cytokines [interleukin-5 (IL-5), IL-7, and IL-12], differential regulation for the mRNA expression of intestinal permeability-related genes (21 downregulated), and disrupted villous architecture, while females showed dose-dependent decreases in inflammatory cytokines [IL-17, IL-5, and macrophage colony-stimulating factor (M-CSF)]. In females, while some intestinal permeability genes were downregulated, the upregulation of other permeability genes suggests a compensatory mechanism to maintain the intestinal barrier function, indicating an overall milder response to TC-ART.DiscussionThese findings suggest that perinatal exposure to TC-ART may have differential impacts on intestinal health, with females exhibiting a more adaptive response compared to males, highlighting the need for sex-specific considerations in evaluating long-term effects of ART.

Despite the highly effective impact of antiretroviral therapy (ART) in reducing mother-to-child transmission of human immunodeficiency virus (HIV), there are concerns of long-term impacts of ART on the health of the offspring. The implications of perinatal exposure to antiviral drugs on the gut bacterial population and metabolic function in the offspring is unclear but may influence health outcomes given the various reported effects of the microbiome in human health. This study aims to gain insight into the potential effect of in utero and lactational exposure to ART on gut microbiota populations and short-chain fatty acids (SCFAs) production in aged rat offspring. Pregnant rats were administered a combination of antiretroviral drugs (abacavir/dolutegravir/lamivudine) at two different dose levels during gestation and throughout lactation, and the fecal bacterial abundance and SCFA levels of the offspring were analyzed when they reached 12 months of age. Our results showed dose-dependent and sex-based differences in fecal microbial abundance at various taxonomic levels. Specifically, we found a decline in Firmicutes in males, and an increase in Actinobacteria among males and females. Furthermore, a sex-specific distribution reorganization of Lactobacillus, Bifidobacterium, and Akkermansia was identified. No significant difference in the concentration of prominent SCFAs and IgA levels were identified. These findings provide preliminary information indicating the need to evaluate perinatal effects of ART more comprehensively on the gut bacterial and metabolic function in future studies, and their potential role in offspring health outcomes.

Behavioral assays are critical in evaluating impacts on nervous system function in rodents due to genetic or environmental factors and are frequently incorporated into regulatory decision-making studies. Despite numerous sources of guidance for such studies, results across behavioral assays are reputed to be highly variable with questionable replicability. Behavioral data obtained from control rats within four contract laboratory studies were used to evaluate replicability across studies, calculate the level of statistical power, and estimate the number of animals required for a specific effect size. For the three behaviors evaluated here (motor activity, acoustic startle response, and learning and memory), control rats from all studies showed the expected pattern of behavior, e.g., open field acclimation, startle habituation, % prepulse inhibition (PPI) over pre-pulse intensities, and acquisition and goal quadrant preference in the Morris Water Maze (MWM). For selected representative individual endpoints, power analyses were conducted to evaluate sample size requirements. Across all endpoints, a drop in power occurred as differences between two groups became smaller. Power analysis of multiple representative endpoints suggested that a sample size of 20 may detect a 30 % effect with 80 % power. Sample size requirements changed with the effect size, and achieving 80 % power with a 20 % effect size generally required a sample size of 30 rats. While the behavioral performance was replicated over the Study Cohorts, power analyses suggested a need for moderation of expectations regarding detectable differences if decisions relied on single endpoints or small effect sizes. Reporting results from a low powered study can have significant and wide-ranging impacts, including undermining confidence in data interpretation, misleading future research, and failing to adhere to the ethical framework of the 3 R's.

The antiretroviral (ARV) drug combination of abacavir sulfate, dolutegravir, and lamivudine [ABC/DTG/3TC; Tri combination Anti-retroviral therapy (TC-ART)] has revolutionized HIV treatment by effectively targeting different stages of viral replication. Despite its therapeutic efficiency for maintaining low viremia in the mother during pregnancy, there are concerns for long-term liabilities in offspring that are indirectly exposed during vulnerable periods of development. The commensal microbiota plays a crucial role in maintaining overall gut health, and disruption of the microbiome is often linked to various extraintestinal effects such as immune dysregulation and inflammation. We recently reported the effects of this drug combination in altering fecal microbiome composition of aged rats perinatally exposed to ABC/DTG/3TC-ART. The fecal microbiome can provide only a snapshot of the composition of microbial community at the end of the digestive tract, which may not reflect the microbial population interacting with ileal mucosa. Thus, the current work reports the effects of this drug combination in the gut mucosa-associated microbiome of the same animals, which showed significant microbial diversity and species richness in high dose exposed female adult offspring, along with dose-dependent changes in Firmicutes/Bacteroidetes ratio. The high dose exposure also showed an increase in opportunistic bacterial species in male animals. Overall, we found that, similar to the fecal microbiome, perinatal exposure to TC-ART led to sex- and dose-dependent alterations in the gut mucosa-associated microbial population in aged rats, suggesting that early life exposure to these drugs may influence gut mucosa-associated immune responses and intestinal permeability.

Cardiovascular (CV) disease in pregnancy is the leading cause of maternal mortality worldwide, often associated with the cardiometabolic remodeling needed for fetal growth. In healthy pregnancies these changes reverse postpartum with no consequence, but improper reverse remodeling increases long-term CV risk. Pregnant patients with HIV are at further risk, but it remains unclear whether this is due to chronic low viremia or administration of antiretrovirals (ART). ART used in pregnancy includes nucleoside reverse transcriptase inhibitors (NRTIs), which have known mitochondrial toxicity, and may impact cardiac function. Here, we tested the hypothesis that NRTI-containing ART negatively impacts the maternal heart. Using rats, we show that pregnancy-associated reverse remodeling was impaired by ART. Molecularly, we found mitochondrial changes in the heart and in the liver, leading to dyslipidemia that collectively affected the heart. Our data suggest caution in the choice of ART prescribed for women with HIV during pregnancy and highlight the importance of studies focusing on maternal health. ### Competing Interest Statement The authors have declared no competing interest.

Triclosan (TCS), a broad-spectrum antibacterial chemical, is detected in human urine, breast milk, amniotic fluid, and feces; however, little is known about its impact on the intestinal microbiome and host mucosal immunity during pregnancy and early development. Pregnant female rats were orally gavaged with TCS from gestation day (GD) 6 to postpartum (PP) day 28. Offspring were administered TCS from postnatal day (PND) 12 to 28. Studies were conducted to assess changes in the intestinal microbial population (16S-rRNA sequencing) and functional analysis of microbial genes in animals exposed to TCS during pregnancy (GD18), and at PP7, PP28 and PND28. Microbial abundance was compared with the amounts of TCS excreted in feces and IgA levels in feces. The results reveal that TCS decreases the abundance of Bacteroidetes and Firmicutes with a significant increase in Proteobacteria. At PND28, total Operational Taxonomic Units (OTUs) were higher in females and showed correlation with the levels of TCS and unbound IgA in feces. The significant increase in Proteobacteria in all TCS-treated rats along with the increased abundance in OTUs that belong to pathogenic bacterial communities could serve as a signature of TCS-induced dysbiosis. In conclusion, TCS can perturb the microbiome, the functional activities of the microbiome, and activate mucosal immunity during pregnancy and early development.

Null hypothesis significance testing is a statistical tool commonly employed throughout laboratory animal research. When experimental results are reported, the reproducibility of the results is of utmost importance. Establishing standard, robust, and adequately powered statistical methodology in the analysis of laboratory animal data is critical to ensure reproducible and valid results. Simulation studies are a reliable method for assessing the power of statistical tests, however, biologists may not be familiar with simulation studies for power despite their efficacy and accessibility. Through an example of simulated Harlan Sprague-Dawley (HSD) rat organ weight data, we highlight the importance of conducting power analyses in laboratory animal research. Using simulations to determine statistical power prior to an experiment is a financially and ethically sound way to validate statistical tests and to help ensure reproducibility of findings in line with the 4R principles of animal welfare.

Two organophosphate esters used as flame retardants and plasticizers, triphenyl phosphate (TPHP) and isopropylated phenyl phosphate (IPP), have been detected in environmental samples around the world. Human exposure primarily occurs via oral ingestion with reported higher concentrations in children. Currently, there are no data to evaluate potential risk from exposure to either TPHP or IPP during fetal development. These short-term perinatal studies in rats provide preliminary toxicity data for TPHP and IPP, including information on transfer to fetus/offspring and across the pup blood-brain barrier. In separate experiments, TPHP or IPP were administered via dosed feed at concentrations 0, 1000, 3000, 10 000, 15 000, or 30 000ppm to time-mated Hsd:Sprague Dawley SD rats from gestation day (GD) 6 through postnatal day (PND) 28; offspring were provided dosed feed at the same concentration as their dam (PND 28-PND 56). TPHP- and IPP-related toxicity resulted in removal of both 30 000ppm groups on GD 12 and 15 000ppm IPP group after parturition. Body weight and organ weights were impacted with exposure in remaining dams. Reproductive performance was perturbed at >= 10 000 ppm TPHP and all IPP exposure groups. In offspring, both TPHP- and IPP-related toxicity was noted in pups at >= 10 000ppm as well as reduction in bodyweights, delays in pubertal endpoints, and/or reduced cholinesterase enzyme activity starting at 1000ppm TPHP or IPP. Preliminary internal dose assessment indicated gestational and lactational transfer following exposure to TPHP or IPP. These findings demonstrate that offspring development is sensitive to 1000ppm TPHP or IPP exposure.

Compromised maternal health leading to maternal seizures can have adverse effects on the healthy development of offspring. This may be the result of inflammation, hypoxia-ischemia, and altered GABA signaling. The current study examined cortical tissue from F2b (2nd litter of the 2nd generation) postnatal day 4 (PND4) offspring of female Harlan SD rats chronically exposed to the seizuregenic compound, 4-Methylimidazole (0, 750, or 2500 ppm 4-MeI). Maternal seizures were evident only at 2500 ppm 4-MeI. GABA related gene expression as examined by qRT-PCR and whole genome microarray showed no indication of disrupted GABA or glutamatergic signaling. Canonical pathway hierarchical clustering and multi-omics combinatory genomic (CNet) plots of differentially expressed genes (DEG) showed alterations in genes associated with regulatory processes of cell development including neuronal differentiation and synaptogenesis. Functional enrichment analysis showed a similarity of cellular processes across the two exposure groups however, the genes comprising each cluster were primarily unique rather than shared and often showed different directionality. A dose-related induction of cytokine signaling was indicated however, pathways associated with individual cytokine signaling were not elevated, suggesting an alternative involvement of cytokine signaling. Pathways related to growth process and cell signaling showed a negative activation supporting an interpretation of disruption or delay in developmental processes at the 2500 ppm 4-MeI exposure level with maternal seizures. Thus, while GABA signaling was not altered as has been observed with maternal seizures, the pattern of DEG suggested a potential for alteration in neuronal network formation.

Thallium is a heavy metal that is known to induce a broad spectrum of adverse health effects in humans including alopecia, neurotoxicity, and mortality following high dose acute poisoning events. Widespread human exposure to thallium may occur via consumption of contaminated drinking water; limited toxicity data are available to evaluate the corresponding public health risk. To address this data gap, the Division of Translational Toxicology conducted short-term toxicity studies of a monovalent thallium salt, thallium (I) sulfate. Thallium (I) sulfate was administered via dosed drinking water to time-mated Sprague Dawley (Hsd:Sprague Dawley® SD®) rats (F0 dams) and their offspring (F1) from gestation day (GD) 6 until up to postnatal day (PND) 28 at concentrations of 0, 3.13, 6.25, 12.5, 25, or 50 mg/L, and adult male and female B6C3F1/N mice for up to 2 weeks at concentrations of 0, 6.25, 12.5, 25, 50, or 100 mg/L. Rat dams in the 50 mg/L exposure group were removed during gestation, and dams and offspring in the 25 mg/L exposure group were removed on or before PND 0 due to overt toxicity. Exposure to thallium (I) sulfate at concentrations ≤ 12.5 mg/L did not impact F0 dam body weights, maintenance of pregnancy, littering parameters, or F1 survival (PND 4–28). However, in F1 pups, exposure to 12.5 mg/L thallium (I) sulfate resulted in decreased body weight gains relative to control rats and onset of whole-body alopecia. Measurement of thallium concentrations in dam plasma, amniotic fluid, fetuses (GD 18), and pup plasma (PND 4) indicated marked maternal transfer of thallium to offspring during gestation and lactation. Mice exposed to 100 mg/L thallium (I) sulfate were removed early due to overt toxicity, and mice exposed to ≥ 25 mg/L exhibited exposure concentration-related decreases in body weight. Lowest-observed-effect levels of 12.5 mg/L (rats) and 25 mg/L (mice) were determined based on the increased incidence of clinical signs of alopecia in F1 rat pups and significantly decreased body weights for both rats and mice.

Parametric statistical tests used to assess body weight changes in rodent experiments assume a normal distribution, and the actual distribution of the rodent body weights is often assumed to be approximately normal. In order for statistical tests to be deemed appropriate without routinely confirming the normal distribution for rodent body weight data, the tests must be powerful enough to detect meaningful changes even when a population deviates from a normal distribution. Here, we present a novel analysis to assess the normality of rodent body weight data for control animals in 1,386 National Toxicology Program (NTP) studies and determined how robust a set of procedures are to detect departures from normality. The distributions of terminal body weight measurements from 90 day and chronic NTP studies were evaluated for normality using graphical and statistical testing methods. The percent of studies with terminal body weights that were not normally distributed in normality tests was typically higher in 90-day studies for Fischer 344/N (F344/N) rats and B6C3F1/N (B6C3F1) mice than Harlan Sprague-Dawley (HSD) rats across all routes of administration evaluated (feed, drinking water, gavage or inhalation). Through simulation studies, the t-test indicated adequate power to detect a difference in body weights in male B6C3F1 mice and F344/N rats in 90-day studies, even under a skew normal distribution. According to these results, common parametric tests display enough power to accurately detect body weight differences from populations not following a normal distribution, confirming the general notion that the study designs are appropriately powered. In addition to providing adequate power, the False Positive Rate (FPR) was controlled around 5% in all simulations. These results suggest that parametric tests are robust enough to give reliable results of body weight analysis in NTP studies where this is an important endpoint. Therefore, parametric testing approaches are appropriate to detect body weight changes in NTP studies when body weight distributions do not deviate too far from normality. Future steps will look at the distributions of non-terminal body weights in chronic studies, organ weights, and other species and strains of rodents.

Background When analyzing fetal defect incidence in laboratory animal studies, correlation in responses within litters (i.e., litter effects) can lead to increased false-positive rates if litter effects are not incorporated into the analysis. Studies of fetal defects require analysis methods that are robust across a broad range of defect types, including those with zero or near-zero incidence rates in control groups. Methods A simulation study compared power and false-positive rates for six approaches across a range of background defect rates and litter size distributions. Statistical methods evaluated included ignoring the litter effect as well as parametric and nonparametric approaches based on litter proportions, generalized linear mixed models (GLMMs), the Rao-Scott Cochran-Armitage (RSCA) trend test, and a modification to the RSCA (mRSCA) introduced here to improve estimation at low background rates. These methods were also applied to a common and a rare defect from two prenatal developmental toxicology studies conducted by the National Toxicology Program (NTP). Results At background defect rates of 1%, the mRSCA and parametric litter proportion methods provided gains in power over the nonparametric litter proportion method, the GLMM method, and the RSCA method. Simulations involving litter loss in high-dose groups showed loss of power for both litter proportion methods. Conclusions The mRSCA test developed here compares favorably with other litter-based approaches and is robust across a range of background defect rates and litter size distributions, making it a practical choice for prenatal developmental toxicology studies involving both common and rare fetal defects.

Due to structural similarity to bisphenol A and lack of safety data, the National Toxicology Program (NTP) is evaluating the potential toxicity of bisphenol AF (BPAF) in rodent models. The current investigation reports the internal exposure data for free (unconjugated BPAF) and total (free and conjugated forms) BPAF during critical stages of development following perinatal dietary exposure in Hsd:Sprague Dawley (R) SD (R) rats to 0 (vehicle control), 338, 1125, and 3750 ppm BPAF from gestation day (GD) 6 to postnatal day (PND) 28. Free and total BPAF concentrations in maternal plasma at GD 18, PND 4, and PND 28 increased with the exposure concentration; free BPAF concentrations were <= 1.61% those of total BPAF demonstrating extensive first pass metabolism of BPAF following dietary exposure in adults. Free and total BPAF were quantified in GD 18 fetuses and PND 4 pups with free concentrations 11.7-53.4% that of corresponding total concentrations. In addition, free concentrations were higher (130-571%) and total concentrations were lower (1.71-7.23%) than corresponding concentrations in dams, demonstrating either preferential transfer of free BPAF and/or inability of fetuses and pups to conjugate BPAF. Free and total concentrations in PND 28 pups were similar to maternal concentrations demonstrating direct exposure of pups via feed and that conjugating enzymes are developed in PND 28 pups. In conclusion, these data demonstrate considerable gestational and lactational transfer of parent aglycone from the mother to offspring. Since the ontogeny of conjugating enzymes in humans is similar to that of rodents, the data from rodent BPAF studies may be useful in predicting human risk from exposure to BPAF.

2,2'-Dimorpholinodiethyl ether (DMDEE) is a specialty amine catalyst used in the production of flexible foams, adhesives and coatings. The potential for occupational exposure to DMDEE is high, but toxicity data are very limited. The objective of this work was to develop a method to quantitate DMDEE in biological matrices to assess gestational and lactational transfer of DMDEE in rats following exposure of dams The method used protein precipitation, followed by removal of phospholipids and analysis of supernatant by ultra-performance liquid chromatography-tandem mass spectrometry. Rat fetuses were homogenized in water prior to protein precipitation and delipidation procedures. The method was evaluated in male Sprague Dawley rat plasma over the concentration range 5 to 1000 ng/mL. The method was linear (r >= 0.99), accurate (mean relative error (RE)<=+/- 11.9%) and precise (relative standard deviation (RSD)<= 2.7%). The mean absolute recovery was 106%. The limit of detection was 0.262 ng/mL. Standards as high as similar to 100,000 ng/mL could be successfully diluted into the calibration range (mean %RE=-14.9; %RSD=0.5). The method was evaluated in Sprague Dawley rat dam plasma, post-natal day 4 pup plasma, gestational day (GD) 18 amniotic fluid and fetal homogenate (mean %RE <=+/- 11.9; %RSD <= 2.3). Concentrations of DMDEE in rat dam plasma, amniotic fluid and fetal homogenate stored for at least 29 days and in pup plasma for at least 18 days at-80 degrees C were within 87.7 to 99.5% of Day 0 concentrations, demonstrating that DMDEE is stable in these matrices. The method was used to quantitate DMDEE in rat plasma, amniotic fluid and fetus samples from a dose range finding toxicology study in which dams were dosed via gavage with DMDEE from GD 6 at doses of 0 (control), 62.5 and 250 mg/kg/day. DMDEE concentration increased with the dose in all matrices examined. The concentration in GD 18 fetuses was almost 2-fold higher than GD 18 dams demonstrating gestational transfer of DMDEE. However, the concentration in post-natal day 4 pup plasma was more than an order of magnitude lower than corresponding dam plasma suggesting less potential for transfer of DMDEE from dams to pups via lactation. There was no significant difference in concentration for male and female pup plasma.