During pregnancy, proper hepatobiliary transport and bile acid synthesis protect the liver from cholestatic injury and regulate the maternal and fetal exposure to bile acids, drugs, and environmental chemicals. The objective of this study was to determine the temporal messenger RNA (mRNA) and protein profiles of uptake and efflux transporters as well as bile acid synthetic and conjugating enzymes in livers from virgin and pregnant mice on gestational days (GD) 7, 11, 14, and 17 and postnatal days (PND) 1, 15, and 30. Compared with virgins, the mRNAs of most transporters were reduced approximately 50% in pregnant dams between GD11 and 17. Western blot and immunofluorescence staining confirmed the downregulation of Mrp3, 6, Bsep, and Ntcp proteins. One day after parturition, the mRNAs of many uptake and efflux hepatobiliary transporters remained low in pregnant mice. By PND30, the mRNAs of all transporters returned to virgin levels. mRNAs of the bile acid synthetic enzymes in the classic pathway, Cyp7a1 and 8b1, increased in pregnant mice, whereas mRNA and protein expression of enzymes in the alternative pathway of bile acid synthesis (Cyp27a1 and 39a1) and conjugating enzymes (Bal and Baat) decreased. Profiles of transporter and bile acid metabolism genes likely result from coordinated downregulation of transcription factor mRNA (CAR, LXR, PXR, PPARα, FXR) in pregnant mice on GD14 and 17. In conclusion, pregnancy caused a global downregulation of most hepatic transporters, which began as early as GD7 for some genes and was maximal by GD14 and 17, and was inversely related to increasing concentrations of circulating 17β-estradiol and progesterone as pregnancy progressed.
pregnancy; transporter; liver; mice; bile acids; nuclear receptors
The ability to effectively monitor gene mutation and micronucleated reticulocyte (MN-RET) frequency in short-term and repeated dosing schedules was investigated using the recently developed flow cytometric Pig-a mutation assay and flow cytometric micronucleus analysis. Eight reference genotoxicants and three presumed nongenotoxic compounds were studied: chlorambucil, melphalan, thiotepa, cyclophosphamide, azathioprine, 2-acetylaminofluorene, hydroxyurea, methyl methanesulfonate, o-anthranilic acid, sulfisoxazole, and sodium chloride. These experiments extend previously published results with seven other chemicals. Male Sprague Dawley rats were treated via gavage for 3 or 28 consecutive days with several dose levels of each chemical up to the maximum tolerated dose. Blood samples were collected at several time points up to day 45 and were analyzed for Pig-a mutation with a dual-labeling method that facilitates mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. An immunomagnetic separation technique was used to increase the efficiency of scoring mutant cells. Blood samples collected on day 4, and day 29 for the 28-day study, were evaluated for MN-RET frequency. The three nongenotoxicants did not induce Pig-a or MN-RET responses. All genotoxicants except hydroxyurea increased the frequency of Pig-a mutant reticulocytes and erythrocytes. Significant increases in MN-RET frequency were observed for each of the genotoxicants at both time points. Whereas the highest Pig-a responses tended to occur in the 28-day studies, when total dose was greatest, the highest induction of MN-RET was observed in the 3-day studies, when dose per day was greatest. There was no clear relationship between the maximal Pig-a response of a given chemical and its corresponding maximal MN-RET response, despite the fact that both endpoints were determined in the same cell lineage. Taken with other previously published results, these data demonstrate the value of integrating Pig-a and micronucleus endpoints into in vivo toxicology studies, thereby providing information about mutagenesis and chromosomal damage in the same animals from which toxicity, toxicokinetics, and metabolism data are obtained.
Pig-a gene; mutation; flow cytometry; micronuclei; genotoxicity; peripheral blood.
Tetramethylenedisulfotetramine (TETS) is a potent convulsant that is considered a chemical threat agent. We characterized TETS as an activator of spontaneous Ca2+ oscillations and electrical burst discharges in mouse hippocampal neuronal cultures at 13–17 days in vitro using FLIPR Fluo-4 fluorescence measurements and extracellular microelectrode array recording. Acute exposure to TETS (≥ 2µM) reversibly altered the pattern of spontaneous neuronal discharges, producing clustered burst firing and an overall increase in discharge frequency. TETS also dramatically affected Ca2+ dynamics causing an immediate but transient elevation of neuronal intracellular Ca2+ followed by decreased frequency of Ca2+ oscillations but greater peak amplitude. The effect on Ca2+ dynamics was similar to that elicited by picrotoxin and bicuculline, supporting the view that TETS acts by inhibiting type A gamma-aminobutyric acid (GABAA) receptor function. The effect of TETS on Ca2+ dynamics requires activation of N-methyl-d-aspartic acid (NMDA) receptors, because the changes induced by TETS were prevented by MK-801 block of NMDA receptors, but not nifedipine block of L-type Ca2+ channels. Pretreatment with the GABAA receptor-positive modulators diazepam and allopregnanolone partially mitigated TETS-induced changes in Ca2+ dynamics. Moreover, low, minimally effective concentrations of diazepam (0.1µM) and allopregnanolone (0.1µM), when administered together, were highly effective in suppressing TETS-induced alterations in Ca2+ dynamics, suggesting that the combination of positive modulators of synaptic and extrasynaptic GABAA receptors may have therapeutic potential. These rapid throughput in vitro assays may assist in the identification of single agents or combinations that have utility in the treatment of TETS intoxication.
Ca2+; oscillations; GABAA receptors; microelectrode array; NMDA receptors; rapid throughput assay; tetramethylenedisulfotetramine.
Methylmercury (MeHg) is an environmental pollutant that biomagnifies throughout the aquatic food chain, thus representing a toxicological concern for humans subsiding on fish for their dietary intake. Although the developing brain is considered the critical target organ of MeHg toxicity, recent evidence indicates that the cardiovascular system may be the most sensitive in adults. However, data on the mechanisms mediating MeHg-induced cardiovascular toxicity are scarce. Based on the close relationship between cardiovascular disease and dyslipidemia, this study was designed to investigate the effects of long-term MeHg exposure on plasma lipid levels in mice, as well as their underlying mechanisms and potential relationships to MeHg-induced neurotoxicity. Our major finding was that long-term MeHg exposure induced dyslipidemia in rodents. Specifically, Swiss and C57BL/6 mice treated for 21 days with a drinking solution of MeHg (40mg/l, ad libitum) diluted in tap water showed increased total and non-HDL plasma cholesterol levels. MeHg-induced hypercholesterolemia was also observed in low-density lipoprotein receptor knockout (LDLr–/–) mice, indicating that this effect was not related to decreased LDLr-mediated cholesterol transport from blood to other tissues. Although the hepatic synthesis of cholesterol was unchanged, significant signs of nephrotoxicity (glomerular shrinkage, tubular vacuolization, and changed urea levels) were observed in MeHg-exposed mice, indicating that the involvement of nephropathy in MeHg-induced lipid dyshomeostasis may not be ruled out. Notably, Probucol (a lipid-lowering drug) prevented the development of hypercholesterolemia when coadministered with MeHg. Finally, hypercholesterolemic LDLr–/– mice were more susceptible to MeHg-induced cerebellar glial activation, suggesting that hypercholesterolemia in itself may pose a risk factor in MeHg-induced neurotoxicity. Overall, based on the strong and graded positive association between total as well as LDL cholesterol and risk of cardiovascular diseases, our data support the concept of MeHg-induced cardiovascular toxicity.
methylmercury; cardiovascular disease; neurotoxicity; low-density lipoprotein receptor; cholesterol; dyslipidemia
Health effects due to environmental exposure to arsenic are a major global health concern. Arsenic has been known to induce carcinogenesis and enhance tumor development via complex and unclear mechanism. Ethanol is also a well-established risk factor for many malignancies. However, little is known about the effects of coexposure to arsenic and ethanol in tumor development. In this study, we investigate the signaling and angiogenic effect of coexposure of arsenic and ethanol on different colon cancer cell lines. Results show that ethanol markedly enhanced arsenic-induced tumor angiogenesis in vitro. These responses are related to intracellular reactive oxygen species (ROS) generation, NADPH oxidase activation, and upregulation of PI3K/Akt and hypoxia-inducible factor 1 alpha (HIF-1α) signaling. We have also found that ethanol increases the arsenic-induced expression and secretion of angiogenic signaling molecules such as vascular endothelial growth factor, which further confirmed the above observation. Antioxidant enzymes inhibited arsenic/ethanol-induced tumor angiogenesis, demonstrating that the responsive signaling pathways of coexposure to arsenic and ethanol are related to ROS generation. We conclude that ethanol is able to enhance arsenic-induced tumor angiogenesis in colorectal cancer cells via the HIF-1α pathway. These results indicate that alcohol consumption should be taken into consideration in the investigation of arsenic-induced carcinogenesis in arsenic-exposed populations.
arsenic; ethanol; reactive oxygen species; hypoxia-inducible factor 1 alpha; tumor angiogenesis
Consumption of deoxynivalenol (DON), a trichothecene mycotoxin known to commonly contaminate grain-based foods, suppresses growth of experimental animals, thus raising concerns over its potential to adversely affect young children. Although this growth impairment is believed to result from anorexia, the initiating mechanisms for appetite suppression remain unknown. Here, we tested the hypothesis that DON induces the release of satiety hormones and that this response corresponds to the toxin’s anorectic action. Acute ip exposure to DON had no effect on plasma glucagon-like peptide-1, leptin, amylin, pancreatic polypeptide, gastric inhibitory peptide, or ghrelin; however, the toxin was found to robustly elevate peptide YY (PYY) and cholecystokinin (CCK). Specifically, ip exposure to DON at 1 and 5mg/kg bw induced PYY by up to 2.5-fold and CCK by up to 4.1-fold. These responses peaked within 15–120min and lasted up to 120min (CCK) and 240min (PPY), corresponding with depressed rates of food intake. Direct administration of exogenous PYY or CCK similarly caused reduced food intake. Food intake experiments using the NPY2 receptor antagonist BIIE0246 and the CCK1A receptor antagonist devazepide, individually, suggested that PYY mediated DON-induced anorexia but CCK did not. Orolingual exposure to DON induced plasma PYY and CCK elevation and anorexia comparable with that observed for ip exposure. Taken together, these findings suggest that PYY might be one critical mediator of DON-induced anorexia and, ultimately, growth suppression.
deoxynivalenol; anorexia; peptide YY; cholecystokinin; mouse
Bisphenol A (BPA) is a high–production volume chemical classified as an environmental estrogen and used primarily in the plastics industry. BPA’s increased usage correlates with rising BPA levels in people and a corresponding increase in the incidence of asthma. Due to limited studies, the contribution of maternal BPA exposure to allergic asthma pathogenesis is unclear. Using two established mouse models of allergic asthma, we examined whether developmental exposure to BPA alters hallmarks of allergic lung inflammation in adult offspring. Pregnant C57BL/6 dams were gavaged with 0, 0.5, 5, 50, or 500 μg BPA/kg/day from gestational day 6 until postnatal day 21. To induce allergic inflammation, adult offspring were mucosally sensitized with inhaled ovalbumin containing low-dose lipopolysaccharide or ip sensitized using ovalbumin with alum followed by ovalbumin aerosol challenge. In the mucosal sensitization model, female offspring that were maternally exposed to ≥ 50 μg BPA/kg/day displayed enhanced airway lymphocytic and lung inflammation, compared with offspring of control dams. Peritoneally sensitized, female offspring exposed to ≤ 50 μg BPA/kg/day presented dampened lung eosinophilia, compared with vehicle controls. Male offspring did not exhibit these differences in either sensitization model. Our data demonstrate that maternal exposure to BPA has subtle and qualitatively different effects on allergic inflammation, which are critically dependent upon route of allergen sensitization and sex. However, these subtle, yet persistent changes due to developmental exposure to BPA did not lead to significant differences in overall airway responsiveness, suggesting that early life exposure to BPA does not exacerbate allergic inflammation into adulthood.
asthma; plastics; developmental exposure; developmental basis of adult disease
Several alkylanilines with structures more complex than toluidines have been associated epidemiologically with human cancer. Their mechanism of action remains largely undetermined, and there is no reported evidence that it replicates that of multicyclic aromatic amines even though the principal metabolic pathways of P450-mediated hydroxylation and phase II conjugation are very similar. As a means to elucidate their mechanisms of action, lethality and mutagenicity in the adenine phosphoribosyltransferase (aprt
+/−) gene induced in several Chinese hamster ovary cell types by 2,6- and 3,5-dimethylaniline (2,6-DMA, 3,5-DMA) and their N- and ring-hydroxyl derivatives (N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP) were assessed. Dose-response relationships were determined in the parental AA8 cell line, its repair-deficient UV5 subclone and other repair-deficient 5P3NAT2 or -proficient 5P3NAT2R9 subclones engineered to express mouse cytochrome P4501A2 (CYP1A2) and human N-acetyltransferase (NAT2), and also in AS52 cells harboring the bacterial guanine-hypoxanthine phosphoribosyltransferase (gpt) gene. Mutations in the gpt gene of AS52 cells were characterized and found to be dominated by G:C to A:T and A:T to G:C transitions. Separately, treatment of AS52 cells with N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP, and 3,5-DMAP led to intracellular production of reactive oxygen species (ROS) for at least 24h after removal of the mutagens in every case. Using the comet assay, DNA strand breaks were observed in a dose-dependent manner in AS52 cells when treated with each of the four N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, and 3,5-DMAP derivatives. Comparative evaluation of the results indicates that the principal mechanism of mutagenic action is likely to be through redox cycling of intracellularly bound aminophenol/quinone imine structures to generate ROS rather than through formation of covalent DNA adducts.
dimethylaniline; alkylanilines; reactive oxygen species; CHO cells; mutations; DNA strand breaks; comet assay
Life-stage-dependent toxicity and dose-dependent toxicokinetics (TK) were evaluated in Sprague Dawley rats following dietary exposure to 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D renal clearance is impacted by dose-dependent saturation of the renal organic anion transporter; thus, this study focused on identifying inflection points of onset of dietary nonlinear TK to inform dose selection decisions for toxicity studies. Male and female rats were fed 2,4-D-fortified diets at doses to 1600 ppm for 4-weeks premating, <2 weeks during mating, and to test day (TD) 71 to parental (P1) males and to P1 females through gestation/lactation to TD 96. F1 offspring were exposed via milk with continuing diet exposure until postnatal day (PND) 35. As assessed by plasma area under the curve for the time-course plasma concentration, nonlinear TK was observed ≥1200 ppm (63mg/kg/day) for P1 males and between 200 and 400 ppm (14–27mg/kg/day) for P1 females. Dam milk and pup plasma levels were higher on lactation day (LD) 14 than LD 4. Relative to P1 adults, 2,4-D levels were higher in dams during late gestation/lactation and postweaning pups (PND 21–35) and coincided with elevated intake of diet/kg body weight. Using conventional maximum tolerated dose (MTD) criteria based on body weight changes for dose selection would have resulted in excessive top doses approximately 2-fold higher than those identified incorporating critical TK data. These data indicate that demonstration of nonlinear TK, if present at dose levels substantially above real-world human exposures, is a key dose selection consideration for improving the human relevance of toxicity studies compared with studies employing conventional MTD dose selection strategies.
2,4-dichlorophenoxyacetic acid; 2,4-D; toxicokinetics; TK; pharmacokinetics; PK; development; perinatal; toxicity; rats.
Rifaximin, a nonsystemic antibiotic that exhibits low gastrointestinal absorption, is a potent agonist of human pregnane X receptor (PXR), which contributes to its therapeutic efficacy in inflammatory bowel disease. To investigate the effects of long-term administration of rifaximin on the liver, PXR-humanized mice were administered rifaximin for 6 months; wild-type and Pxr-null mice were treated in parallel as controls. Histological analysis revealed time-dependent intense hepatocellular fatty degeneration and increased hepatic triglycerides in PXR-humanized mice and not in wild-type and Pxr-null mice. After long-term treatment, PXR target genes were induced in small intestine and liver, with significant up-regulation in the expression of hepatic genes related to triglyceride synthesis and lipid accumulation. However, no significant hepatic accumulation of rifaximin was found, even after 6 months of treatment, in PXR-humanized mice. Genes in the small intestine that are involved in the uptake of fatty acids and triglycerides were induced along with increased triglyceride accumulation in intestinal epithelial cells of PXR-humanized mice; this was not observed in wild-type and Pxr-null mice. These findings suggest that long-term administration of rifaximin could lead to PXR-dependent hepatocellular fatty degeneration as a result of activation of genes involved in lipid uptake, thus indicating a potential adverse effect of rifaximin on liver function after long-term exposure.
rifaximin; hepatotoxicity; human pregnane X receptor; steatosis
We have previously demonstrated a role for the aryl hydrocarbon receptor (AHR) in the attenuation of the cholesterol biosynthesis pathway. This regulation did not require that the AHR binds to its cognate response element. Based on these observations and other reports depicting a role for AHR in lipid metabolism, we chose to investigate the involvement of the receptor in the regulation of the fatty acid synthesis pathway in mice and humans. For this purpose, C57BL/6J, liver-specific transgenic DRE-binding mutant AhR (A78D-Ahr
fx/fx) and Cre
fx/fx mice were treated with an AHR ligand, and hepatic mRNA expression levels of key fatty acid genes (e.g., Acaca, Fasn, Scd1) were measured. The basal levels of those genes were also compared between C57BL6/J and hepatic AHR-deficient mice, as well as between Ah
b and Ah
d congenic mice. To extend these results to humans, fatty acid gene expression in human cells were compared with AHR-silenced cells. In addition, primary human hepatocytes were treated with an AHR ligand to assess alterations in gene expression and fatty acid synthesis. These studies indicated that the AHR constitutively attenuates the expression of key fatty acid synthesis genes in the absence of binding to its cognate response element. In addition, activation of AHR led to further repression of the expression of these genes and a decrease in overall fatty acid synthesis and secretion in human hepatocytes. Based on our results, we can conclude that increased AHR activity represses fatty acid synthesis, suggesting it may be a future therapeutic target.
AHR; Ah receptor; fatty acids; DRE
For over 15 years, reproductive toxicologists have explored the physiological outcomes and mechanism of fetal phthalate exposure to determine the risk posed to human male reproductive health. This review examines the fetal male reproductive system response to phthalate exposure across species including rat, mouse, and human, with emphasis on the testis. In the rat, in utero phthalate exposure causes male reproductive tract malformations, in large part, by targeting the testis and inhibiting fetal Leydig cell hormone production. Despite mouse phthalate pharmacokinetics being similar to the rat, inhibition of fetal Leydig cell hormone synthesis is not observed in the mouse. The species-specific differences in testicular response following in utero phthalate exposure and the discordant reaction of the rodent fetal testis when exposed to phthalates ex vivo versus in vivo have made determining risk to humans difficult, yet critically important. The recent use of fetal testis xenotransplants to study phthalate toxicity suggests that the human fetal testis responds like the mouse fetal testis; it appears refractory to phthalate-induced inhibition of testosterone production. Although this result is unfulfilling from the perspective of identifying environmental contributions to human reproductive maldevelopment, it has important implications for phthalate risk assessment.
phthalates; endocrine disruptors; testicular dysgenesis syndrome; Leydig; fetal testis
Many comparative analyses of toxicity assume that the potency of a test chemical relative to a reference chemical is constant, but employing such a restrictive assumption uncritically may generate misleading conclusions. Recent efforts to characterize non-constant relative potency rely on relative potency functions and estimate them secondarily after fitting dose-response models for the test and reference chemicals. We study an alternative approach of specifying a relative potency model a priori and estimating it directly using the dose-response data from both chemicals. We consider a power function in dose as a relative potency model and find that it keeps the two chemicals’ dose-response functions within the same family of models for families typically used in toxicology. When differences in the response limits for the test and reference chemicals are attributable to the chemicals themselves, the older two-stage approach is the more convenient. When differences in response limits are attributable to other features of the experimental protocol or when response limits do not differ, the direct approach is straightforward to apply with nonlinear regression methods and simplifies calculation of simultaneous confidence bands. We illustrate the proposed approach using Hill models with dose-response data from U.S. National Toxicology Program bioassays. Though not universally applicable, this method of estimating relative potency functions directly can be profitably applied to a broad family of dose-response models commonly used in toxicology.
dose response; Hill model; PeCDF; relative potency; TCDD
MicroRNAs (miRNAs) are endogenous noncoding RNA molecules that are involved in post-transcriptional gene silencing. Using global miRNA expression profiling, we found miR-21, -155, and 18a to be highly upregulated in rat kidneys following tubular injury induced by ischemia/reperfusion (I/R) or gentamicin administration. Mir-21 and -155 also showed decreased expression patterns in blood and urinary supernatants in both models of kidney injury. Furthermore, urinary levels of miR-21 increased 1.2-fold in patients with clinical diagnosis of acute kidney injury (AKI) (n = 22) as compared with healthy volunteers (n = 25) (p < 0.05), and miR-155 decreased 1.5-fold in patients with AKI (p < 0.01). We identified 29 messenger RNA core targets of these 3 miRNAs using the context likelihood of relatedness algorithm and found these predicted gene targets to be highly enriched for genes associated with apoptosis or cell proliferation. Taken together, these results suggest that miRNA-21 and -155 could potentially serve as translational biomarkers for detection of AKI and may play a critical role in the pathogenesis of kidney injury and tissue repair process.
MicroRNAs; kidney; biomarker; ischemia/reperfusion injury; nephrotoxicity.
A crucial period for the development of the immune system occurs in utero. This results in a high fetal vulnerability to immunotoxic exposure, and indeed, immunotoxic effects have been reported, demonstrating negative effects on immune-related health outcomes and immune functionality. Within the NewGeneris cohort BraMat, a subcohort of the Norwegian Mother and Child Cohort Study (MoBa), immunotoxicity was demonstrated for polychlorinated biphenyls and dioxins, showing associations between estimated maternal intake levels and reduced measles vaccination responses in the offspring at the age of 3. The present study aimed to investigate this link at the transcriptomic level within the same BraMat cohort. To this end, whole-genome gene expression in cord blood was investigated and found to be associated with maternal Food Frequency Questionnaires–derived exposure estimates and with vaccination responses in children at 3 years of age. Because the literature reports gender specificity in the innate, humoral, and cell-mediated responses to viral vaccines, separate analysis for males and females was conducted. Separate gene sets for male and female neonates were identified, comprising genes significantly correlating with both 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and polychlorinated biphenyls (PCB) exposure and with measles vaccination response. Noteworthy, genes correlating negatively with exposure in general show positive correlations with antibody levels and vice versa. For both sexes, these included immune-related genes, suggesting immunosuppressive effects of maternal exposure to TCDD and PCB at the transcriptomic level in neonates in relation to measles vaccination response 3 years later.
immunotoxicology; toxicogenomics; newborns; maternal exposure; TCDD; PCB.
Environmental exposure to arsenic, especially the trivalent inorganic form (As3+), has been linked to human cancers in addition to a number of other diseases including skin lesions, cardiovascular disorders, neuropathy, and internal organ injury. In the present study, we describe a novel signaling axis of the c-Jun NH2 kinase (JNK) and signal transducer and activator of transcription 3 (Stat3) and its involvement in As3+-induced Akt activation in human bronchial epithelial cells. As3+ activates JNK and induces phosphorylation of the Stat3 at serine 727 (S727) in a dose- and time-dependent manner, which occurred concomitantly with Akt activation. Disruption of the JNK signaling pathway by treatment with the JNK inhibitor SP600125, siRNA knockdown of JNK, or genetic deficiency of the JNK1 or JNK2 gene abrogated As3+-induced S727 phosphorylation of Stat3, Akt activation, and the consequent release of vascular endothelial growth factor (VEGF) and migration of the cells. Similarly, pretreatment of the cells with Stat3 inhibitor or Stat3 siRNA prevented Akt activation and VEGF release from the cells in response to As3+ treatment. Taken together, these data revealed a new signaling mechanism that might be pivotal in As3+-induced malignant transformation of the cells by linking the key stress signaling pathway, JNK, to the activation of Stat3 and the carcinogenic kinase, Akt.
2,4-Dichlorophenoxyacetic acid (2,4-D) was assessed for systemic toxicity, reproductive toxicity, developmental neurotoxicity (DNT), developmental immunotoxicity (DIT), and endocrine toxicity. CD rats (27/sex/dose) were exposed to 0, 100, 300, 600 (female), or 800 (male) ppm 2,4-D in diet. Nonlinear toxicokinetic behavior was shown at high doses; the renal clearance saturation threshold for 2,4-D was exceeded markedly in females and slightly exceeded in males. Exposure was 4 weeks premating, 7 weeks postmating for P1 males and through lactation for P1 females. F1 offspring were examined for survival and development, and at weaning, pups were divided in cohorts, by sex and dose, and by systemic toxicity (10), DNT (10), DIT (20), and reproductive toxicity (≥ 23). Remaining weanlings were evaluated for systemic toxicity and neuropathology (10–12). Body weight decreased during lactation in high-dose P1 females and in F1 pups. Kidney was the primary target organ, with slight degeneration of proximal convoluted tubules observed in high-dose P1 males and in high-dose F1 males and females. A slight intergenerational difference in kidney toxicity was attributed to increased intake of 2,4-D in F1 offspring. Decreased weanling testes weights and delayed preputial separation in F1 males were attributed to decreased body weights. Endocrine-related effects were limited to slight thyroid hormone changes and adaptive histopathology in high-dose GD 17 dams seen only at a nonlinear toxicokinetic dose. 2,4-D did not cause reproductive toxicity, DNT, or DIT. The “No Observed Adverse Effect Level” for systemic toxicity was 300 ppm in both males (16.6mg/kg/day) and females (20.6mg/kg/day), which is approximately 6700- to 93 000-fold higher than that reported for 2,4-D exposures in human biomonitoring studies.
2,4-D; extended one-generation reproductive toxicity study; EOGRTS; endocrine; androgen; estrogen; thyroid; developmental immunotoxicity; developmental neurotoxicity; reproductive toxicity; systemic toxicity; kinetically derived maximum dose; KMD; toxicokinetics.
The gaseous olefin ethylene (ET) is metabolized in mammals to the carcinogenic epoxide ethylene oxide (EO). Although ET is the largest volume organic chemical worldwide, the EO burden in ET-exposed humans is still uncertain, and only limited data are available on the EO burden in ET-exposed rodents. Therefore, EO was quantified in blood of mice, rats, or 4 volunteers that were exposed once to constant atmospheric ET concentrations of between 1 and 10 000 ppm (rodents) or 5 and 50 ppm (humans). Both the compounds were determined by gas chromatography. At ET concentrations of between 1 and 10 000 ppm, areas under the concentration-time curves of EO in blood (µmol × h/l) ranged from 0.039 to 3.62 in mice and from 0.086 to 11.6 in rats. At ET concentrations ≤ 30 ppm, EO concentrations in blood were 8.7-fold higher in rats and 3.9-fold higher in mice than that in the volunteer with the highest EO burdens. Based on measured EO concentrations, levels of EO adducts to hemoglobin and lymphocyte DNA were calculated for diverse ET concentrations and compared with published adduct levels. For given ET exposure concentrations, there were good agreements between calculated and measured levels of adducts to hemoglobin in rats and humans and to DNA in rats and mice. Reported hemoglobin adduct levels in mice were higher than calculated ones. Furthermore, information is given on species-specific background adduct levels. In summary, the study provides most relevant data for an improved assessment of the human health risk from exposure to ET.
ethylene; ethylene oxide; mouse; rat; human; blood; hemoglobin adducts; DNA adducts.
Some clinicians assess the efficacy of pralidoxime in organophosphorus (OP) poisoned patients by measuring reactivation of butyrylcholinesterase (BuChE). However, the degree of BuChE inhibition varies by OP insecticide, and it is unclear how well oximes reactivate BuChE in vivo. We aimed to assess the usefulness of BuChE activity to monitor pralidoxime treatment by studying its reactivation after pralidoxime administration to patients with laboratory-proven World Health Organization (WHO) class II OP insecticide poisoning. Patient data were derived from 2 studies, a cohort study (using a bolus treatment of 1g pralidoxime chloride) and a randomized controlled trial (RCT) (comparing 2g pralidoxime over 20min, followed by an infusion of 0.5g/h, with placebo). Two grams of pralidoxime variably reactivated BuChE in patients poisoned by 2 diethyl OP insecticides, chlorpyrifos and quinalphos; however, unlike acetylcholinesterase reactivation, this reactivation was not sustained. It did not reactivate BuChE inhibited by the dimethyl OPs dimethoate or fenthion. The 1-g dose produced no reactivation. Pralidoxime produced variable reactivation of BuChE in WHO class II OP-poisoned patients according to the pralidoxime dose administered, OP ingested, and individual patient. The use of BuChE assays for monitoring the effect of pralidoxime treatment is unlikely to be clinically useful.
organophosphorus insecticides; butyrylcholinest erase; pralidoxime; marker; human poisoning.
1,3-Butadiene (BD) is a smoke component selected by the World Health Organization (WHO) study group on Tobacco Product Regulation (TobReg) for mandated lowering. We examined the tobacco smoke–related health effects induced by BD and possible health impacts of risk reduction strategies. BD levels in mainstream smoke (MSS) from international and Canadian cigarettes and environmental tobacco smoke (ETS) were derived from scientific journals and international government reports. Dose-response analyses from toxicity studies from government reports were evaluated and the most sensitive cancer and noncancer endpoints were selected. The risks were evaluated by taking the ratio (margin of exposure, MOE) from the most sensitive toxicity endpoint and appropriate exposure estimates for BD in MSS and ETS. BD is a good choice for lowering given that MSS and ETS were at levels for cancer (leukemia) and noncancer (ovarian atrophy) risks, and the risks can be significantly lowered when lowering the BD concentrations in smoke. Several risk reduction strategies were analyzed including a maximum level of 125% of the median BD value per milligram nicotine obtained from international brands as recommended by the WHO TobReg, tobacco substitute sheets, dual and triple carbon filters, and polymer-derived carbon. The use of tobacco substitute sheet with a polymer-derived carbon filter resulted in the most significant change in risk for cancer and noncancer effects. Our results demonstrate that MOE analysis might be a practical way to assess the impact of risk reduction strategies on human health in the future.
1,3-butadiene; risk reduction strategies; smoke components; mandated lowering; margin of exposure.
A comprehensive analysis was performed to investigate how inhibition of the human bile salt export pump (BSEP/ABCB11) relates to clinically observed drug-induced liver injury (DILI). Inhibition of taurocholate (TA) transport was investigated in BSEP membrane vesicles for a data set of 250 compounds, and 86 BSEP inhibitors were identified. Structure-activity modeling identified BSEP inhibition to correlate strongly with compound lipophilicity, whereas positive molecular charge was associated with a lack of inhibition. All approved drugs in the data set (n = 182) were categorized according to DILI warnings in drug labels issued by the Food and Drug Administration, and a strong correlation between BSEP inhibition and DILI was identified. As many as 38 of the 61 identified BSEP inhibitors were associated with severe DILI, including 9 drugs not previously linked to BSEP inhibition. Further, among the tested compounds, every second drug associated with severe DILI was a BSEP inhibitor. Finally, sandwich-cultured human hepatocytes (SCHH) were used to investigate the relationship between BSEP inhibition, TA transport, and clinically observed DILI in detail. BSEP inhibitors associated with severe DILI greatly reduced the TA canalicular efflux, whereas BSEP inhibitors with less severe or no DILI resulted in weak or no reduction of TA efflux in SCHH. This distinction illustrates the usefulness of SCHH in refined analysis of BSEP inhibition. In conclusion, BSEP inhibition in membrane vesicles was found to correlate to DILI severity, and altered disposition of TA in SCHH was shown to separate BSEP inhibitors associated with severe DILI from those with no or mild DILI.
biliary excretion; drug induced liver injury; hepatocytes; in vitro and alternatives; disposition; risk assessment; alternatives to animal testing; predictive toxicology.
In Japan, people using water from the well contaminated with high-level arsenic developed neurological, mostly cerebellar, symptoms, where diphenylarsinic acid (DPAA) was a major compound. Here, we investigated the adverse effects of developmental exposure to 20mg/l DPAA in drinking water (early period [0–6 weeks of age] and/or late period [7–12]) on behavior and cerebellar development in male rats. In the open field test at 6 weeks of age, early exposure to DPAA significantly increased exploratory behaviors. At 12 weeks of age, late exposure to DPAA similarly increased exploratory behavior independent of the early exposure although a 6-week recovery from DPAA could reverse that change. In the passive avoidance test at 6 weeks of age, early exposure to DPAA significantly decreased the avoidance performance. Even at 12 weeks of age, early exposure to DPAA significantly decreased the test performance, which was independent of the late exposure to DPAA. These results suggest that the DPAA-induced increase in exploratory behavior is transient, whereas the DPAA-induced impairment of passive avoidance is long lasting. At 6 weeks of age, early exposure to DPAA significantly reduced the concentration of cerebellar total glutathione. At 12 weeks of age, late, but not early, exposure to DPAA also significantly reduced the concentration of cerebellar glutathione, which might be a primary cause of oxidative stress. Early exposure to DPAA induced late-onset suppressed expression of NMDAR1 and PSD95 protein at 12 weeks of age, indicating impaired glutamatergic system in the cerebellum of rats developmentally exposed to DPAA.
diphenylarsinic acid; rat; cerebellum; glutathione.
The aryl hydrocarbon receptor (AHR) is a transcription factor that responds to diverse ligands and plays a critical role in toxicology, immune function, and cardiovascular physiology. The structural basis of the AHR for ligand promiscuity and preferences is critical for understanding AHR function. Based on the structure of a closely related protein HIF2α, we modeled the AHR ligand binding domain (LBD) bound to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene (BaP) and identified residues that control ligand preferences by shape and H-bond potential. Mutations to these residues, particularly Q377 and G298, resulted in robust and opposite changes in the potency of TCDD and BaP and up to a 20-fold change in the ratio of TCDD/BaP efficacy. The model also revealed a flexible “belt” structure; molecular dynamic (MD) simulation suggested that the “belt” and several other structural elements in the AHR-LBD are more flexible than HIF2α and likely contribute to ligand promiscuity. Molecular docking of TCDD congeners to a model of human AHR-LBD ranks their binding affinity similar to experimental ranking of their toxicity. Our study reveals key structural basis for prediction of toxicity and understanding the AHR signaling through diverse ligands.
dioxin; Ah receptor; structural modeling; structural determinants; ligand preferences; ligand tolerance.
Editor's Highlight: Melamine and cyanuric acid were added to pet food in the United States and baby formula in China to boost apparent protein content, resulting in widespread poisoning that was characterized by renal toxicity. The research conducted by the Goering group in this issue demonstrated that renal toxicity could be monitored by the analysis of urinary RPA-1 (distal tubule and collecting duct injury biomarker). This represents a novel and valuable biomarker for the noninvasive monitoring of obstructive nephropathy associated with melamine-cyanuric acid exposure.
Oral coexposure of rats to melamine (MEL) and cyanuric acid (CYA) results in a dose-dependent increase in the formation of MEL-CYA crystals in the kidney. The aim of this study was to determine if urinary biomarkers of acute kidney injury could be used to noninvasively detect renal damage associated with crystal formation in the kidneys of MEL- and CYA-exposed rats. Urine was obtained on days 0 (predose), 2, 4, 14, and 28 from male and female Fischer 344 rats fed a diet supplemented with 0, 120, 180, or 240 ppm each of MEL and CYA. A number of urinary protein biomarkers (kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, osteopontin, albumin, alpha-GST, GST-Yb1, renal papillary antigen 1 [RPA-1], and clusterin) were measured using a multiplex assay system. The results showed that RPA-1 (distal tubule and collecting duct injury biomarker) was elevated on day 28 at the 120 ppm dose and higher in male rats and at the 180 ppm dose and higher in female rats; however, other urinary protein biomarkers were significantly elevated only at the 240 ppm dose. Significant elevation in blood urea nitrogen and serum creatinine levels, and severe renal damage evidenced by histopathology, were observed after 28 days of exposure to the highest dose, despite the fact that MEL-CYA crystals were observable at the 120 and 180 ppm doses. These data indicate that RPA-1 may serve as a noninvasive urinary biomarker for the detection and monitoring of obstructive nephropathy associated with MEL-CYA exposure.
kidney injury; obstructive nephropathy; nephrotoxicity biomarker; melamine; cyanuric acid
Diisocyanates are a common cause of occupational asthma, but risk factors are not well defined. A case-control study was conducted to investigate whether genetic variants of antioxidant defense genes, glutathione S-transferases (GSTM1, GSTT1, GSTM3, GSTP1), manganese superoxide dismutase (SOD2), and microsomal epoxide hydrolase (EPHX1) are associated with increased susceptibility to diisocyanate-induced asthma (DA). The main study population consisted of 353 Caucasian French-Canadians from among a larger sample of 410 diisocyanate-exposed workers in three groups: workers with specific inhalation challenge (SIC) confirmed DA (DA+, n = 95); symptomatic diisocyanate workers with a negative SIC (DA−, n = 116); and asymptomatic exposed workers (AW, n = 142). Genotyping was performed on genomic DNA, using a 5′-nuclease PCR assay. The SOD2 rs4880, GSTP1 rs1695, and EPHX1 rs2740171 variants were significantly associated with DA in both univariate and multivariate analyses. In the first logistic regression model comparing DA+ and DA− groups, SOD2 rs4880, GSTM1 (null), GSTP1 rs762803, and EPHX1 rs2854450 variants were associated with DA (p = 0.004, p = 0.047, p = 0.021, p <0.001, respectively). Genotype combinations GSTT1*GSTP1 rs762803, GSTM1*EPHX1 rs2854450, EPHX1 rs2740168*EPHX1 rs1051741, and GSTP1 rs762803*EPHX1 rs2740168 were also associated with DA in this model (p = 0.027, p = 0.002, p = 0.045, p = 0.044, respectively). The GSTP1 rs1695 and EPHX1 rs1051741 and rs2740171 variants showed an association with DA in the second model comparing DA+ and AW groups (p = 0.040, p = 0.019, p = 0.002, respectively). The GSTM3 rs110913*EPHX1 rs1051741 genotype combination was also associated with DA under this model (p = 0.042). The results suggest that variations in SOD2, GST, and EPHX1 genes and their interactions contribute to DA susceptibility.
diisocyanates; occupational asthma; antioxidant; genetics; single-nucleotide polymorphism.