•Perinatal exposure to brominated furans impairs behavior in adult and infant mice.•Adult exploratory behavior and infant calling can be useful behavioral endpoints.•The toxic equivalent factor ...concept can be applied to brominated dioxins/furans.
Polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/DFs) have been unintentionally produced and emitted from the lifecycle of products containing brominated flame retardants, such as polybrominated diphenyl ether, which is suspected to cause developmental neurotoxicity (DNT). Although it is plausible that PBDD/DFs can also induce DNT, information regarding their neurotoxic potential is currently limited. Hence, in the present study, we examined the effects of in utero and lactational exposure to brominated dibenzofurans on infant and adult offspring behavior to understand the mechanism of PBDD/DFs toxicity and detect effective behavioral endpoints in DNT assessment. We analyzed the behavior of mouse offspring born to dams administered 2,3,7,8-tetrabromodibenzofuran (2,3,7,8-TeBDF; dose of 0, 9, or 45 μg/kg) or 2,3,8-tribromodibenzofuran (2,3,8-TrBDF; dose of 0, 75.6, or 378 μg/kg) on gestational day 12.5. In mouse offspring born to dams exposed to 2,3,7,8-TeBDF, the exploratory behavior in a novel environment in adulthood and ultrasonic vocalization (USV) during infancy were significantly reduced. Additionally, AhR-target genes, such as Cyp1a1, were induced in the liver of 2,3,7,8-TeBDF-exposed offspring in a dose-dependent manner. Conversely, no significant changes in the infant and adult behaviors and expression level of AhR-target genes were observed in the 2,3,8-TrBDF-exposed offspring. These results suggest that 2,3,7,8-TeBDF can induce DNT and that the analysis of exploratory behavior in a novel environment and USV may be useful endpoints to assess DNT of dioxin-related substances.
Benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) is widely used as sunscreen for protection of human skin and hair from damage by ultraviolet (UV) radiation. In this study, we examined the ...metabolism of BP-3 by rat and human liver microsomes, and the estrogenic and anti-androgenic activities of the metabolites. When BP-3 was incubated with rat liver microsomes in the presence of NADPH, 2,4,5-trihydroxybenzophenone (2,4,5-triOH BP) and 3-hydroxylated BP-3 (3-OH BP-3) were newly identified as metabolites, together with previously detected metabolites 5-hydroxylated BP-3 (5-OH BP-3), a 4-desmethylated metabolite (2,4-diOH BP) and 2,3,4-trihydroxybenzophenone (2,3,4-triOH BP). In studies with recombinant rat cytochrome P450, 3-OH BP-3 and 2,4,5-triOH BP were mainly formed by CYP1A1. BP-3 was also metabolized by human liver microsomes and CYP isoforms. In estrogen reporter (ER) assays using estrogen-responsive CHO cells, 2,4-diOH BP exhibited stronger estrogenic activity, 2,3,4-triOH BP exhibited similar activity, and 5-OH BP-3, 2,4,5-triOH BP and 3-OH BP-3 showed lower activity as compared to BP-3. Structural requirements for activity were investigated in a series of 14 BP-3 derivatives. When BP-3 was incubated with liver microsomes from untreated rats or phenobarbital-, 3-methylcholanthrene-, or acetone-treated rats in the presence of NADPH, estrogenic activity was increased. However, liver microsomes from dexamethasone-treated rats showed decreased estrogenic activity due to formation of inactive 5-OH BP-3 and reduced formation of active 2,4-diOH BP. Anti-androgenic activity of BP-3 was decreased after incubation with liver microsomes.
•Metabolic modification of the endocrine-disrupting activity of BP-3 was examined.•2,4,5-TriOH BP and 3-OH BP-3 were identified as new BP-3 metabolites.•2,4-DiOH BP and 2,3,4-triOH BP exhibited high or similar estrogenic activities.•Estrogenic activity of BP-3 was enhanced by incubation with rat liver microsomes.•Structural requirements for the activities of BP-3 derivatives were demonstrated.
Polybrominated dibenzo‐p‐dioxins and dibenzofurans (PBDD/DFs) are byproducts of brominated flame retardants and can cause adverse health effects. Although exposure to polychlorinated (PC) DD/DFs ...induces toxic effects, including liver injury and neurobehavioral disorder, little is known about toxicities associated with PBDD/DF exposure. Thus, we examined effects of perinatal exposure to brominated congener on the infant mouse. Gene expression in several organs, such as the liver and brain, was analyzed in mouse offspring born to dams administered 2,3,7,8‐tetrabromodibenzofuran (TBDF; 9 or 45 μg/kg body weight) or 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD; 3 μg/kg body weight) on gestational day 12.5. An increase in liver size was observed in TBDF‐ or TCDD‐exposed offspring in infancy. Gene microarray analysis revealed that 163 and 36 genes were markedly upregulated and downregulated, respectively, in the liver of TBDF‐exposed mice compared with those in vehicle‐treated mice on postnatal day (PND) 5. Significant increases in Cyp1a1, Cyp1a2, Fmo3, and Pnliprp1 and decreases in Tff3, Ocstamp, Kcnk16, and Lgals2 mRNA levels in TBDF‐exposed offspring on PNDs 5 and 12 were confirmed by quantitative PCR. In particular, a significant reduction in Tff3 mRNA in the liver, but not in the brain, small intestine, colon, and kidney, was observed in offspring perinatally exposed to TBDF or TCDD. Ultrasonic calls of TBDF‐ or TCDD‐exposed offspring on PNDs 3–5 were impaired. Taken together, perinatal exposure to polyhalogenated dioxin/furan congeners disrupts gene expression patterns in the liver and ultrasonic calling during infancy. These results suggest that liver injury may contribute to neurobehavioral disorder.
Information regarding the toxicity of polybrominated dibenzofurans is currently limited. We revealed that perinatal exposure to 2,3,7,8‐tetrabromodibenzofuran (TBDF) disrupted gene expression patterns in the infant mouse liver and induced a liver‐specific decrease in Tff3 mRNA expression. A reduction in liver Tff3 mRNA expression was also observed in infant mice exposed to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). Ultrasonic calls were impaired by TBDF or TCDD exposure. Collectively, perinatal exposure to polyhalogenated dioxin/furan congeners induces liver injury and neurobehavioral disorder during developmental period.
World Health Organization toxic equivalency factors (WHO-TEFs) are recommended for risk management of brominated dioxins in aquatic environments because limited information is available on their ...toxicity to fish. To validate this approach, we obtained the relative potencies of polybrominated dibenzo-p-dioxins and polybrominated dibenzofurans and mixed-halogenated furans (PXDF, X = Cl/Br) against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) based on their toxicity to the early-life stage of Japanese medaka (Oryzias latipes). 2,3,7,8-substituted brominated dibenzofurans caused typical dioxin exposure effects, such as blue-sac disease. The TCDD-relative potency factors (REPs) of test substances were calculated based on the concentrations in water and eggs that caused 20% lethality on day 28 post-fertilization, and were in the order of: 2-chloro-3,7,8-tribromodibenzofuran (REPwater 3.3, REPegg 4.6) > 2,3,7,8-tetrabromodibenzofuran (0.85, 0.92) > 2,3,4,7,8-pentabromodibenzofuran (0.053, 0.55) > 1,2,3,7,8-pentabromodibenzofuran (0.0091, 0.19). The transfer rate from water to eggs was lower for pentabrominated furans than tetrabrominated congeners, and was expected to decrease with the log Kow of the test substance. Although the REPegg value can be used to compare the toxicity potential of brominated dioxins, REPwater may be more suitable for environmental risk assessment because the uptake potential of these compounds from water should be considered. This study is the first to report higher toxicity of a PXDF congener compared with TCDD in vivo, further investigations of the toxicity of mixed-halogenated dioxins and environmental behavior are necessary for environmental risk assessment.
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•Effects of brominated dioxins in the early-life stage of Japanese medaka were examined.•2,3,7,8-halogenated dibenzofurans caused typical effects of dioxin-like compounds.•The uptake rate of brominated dioxins needs to be considered in risk assessment.•Some mixed-halogenated dioxins may have higher toxicity than TCDD.
To develop novel nonallergenic pyrazolone analgesics, we synthesized a series of compounds in which position 1 of the pyrazolone ring was substituted in place of the original methyl group in order to ...block the formation of allergenic metabolites via N-dealkylation. These pyrazolone analogues were found to show as potent an antipyretic and analgesic effect as antipyrine (AT). In an examination of allergenicity, AT induced a typical skin reaction in guinea pigs, whereas the pyrazolone analogues were inactive. When AT was administered (po) to rats, norantipyrine (NORA) as an active metabolite was detected in the urine, whereas similar administration of the pyrazolone analogues did not afford NORA. We conclude that these novel pyrazolone analogues were nonallergenic because they were not converted to allergenic metabolites in vivo. Because these compounds retain the antipyretic and analgesic activities of AT, they are considered to be promising candidates for nonallergenic antipyretic analgesics.
•Hepatic T3-responsive genes were identified in neonatal rats for the first time.•Slc25a25 expression was upregulated particularly in neonatal rats by low doses of T3.•Hdc expression was ...downregulated particularly in neonatal rats by low doses of T3.
There have been many concerns about the possible adverse effects of thyroid hormone-disrupting chemicals in the environment. Because thyroid hormones are essential for regulating the growth and differentiation of many tissues, disruption of thyroid hormones during the neonatal period of an organism might lead to permanent effects on that organism. We postulated that there are target genes that are sensitive to thyroid hormones particularly during the neonatal period and that would thus be susceptible to thyroid hormone-disrupting chemicals. Global gene expression analysis was used to identify these genes in the liver of rat neonates. The changes in hepatic gene expression were examined 24 h after administering 1.0, 10, and 100 ng/g body weight (bw) triiodothyronine (T3) to male rats on postnatal day 3. Thirteen upregulated and four downregulated genes were identified in the neonatal liver. Among these, Pdp2 and Slc25a25 were found to be upregulated and more sensitive to T3 than the others, whereas Cyp7b1 and Hdc were found to be downregulated even at the lowest dose of 1.0 ng/g bw T3. Interestingly, when the responses of gene expression to T3 were examined in adult rats (8-week old), one-third of them did not respond to T3. The environmental chemicals with thyroid hormone-like activity, hydroxylated polybrominated diphenyl ethers, were then administered to neonatal rats to examine the effects on expression of the identified genes. The results showed that these chemicals were indeed capable of changing the expression of Slc25a25 and Hdc. Our results demonstrated a series of hepatic T3-responsive genes that are more sensitive to hormones during the neonatal period than during adulthood. These genes might be the potential targets of thyroid hormone-disrupting chemicals in newborns.
Indoor dust is a sink for many kinds of pollutants, including flame retardants (FRs), plasticizers, and their contaminants and degradation products. These pollutants can be migrated to indoor dust ...from household items such as televisions and computers. To reveal high-priority end points of and contaminant candidates in indoor dust, using CALUX reporter gene assays based on human osteosarcoma (U2OS) cell lines, we evaluated and characterized the endocrine-disrupting potencies of crude extracts of indoor dust collected from Japan (n = 8), the United States (n = 21), Vietnam (n = 10), the Philippines (n = 17), and Indonesia (n = 10) and for 23 selected FRs. The CALUX reporter gene assays used were specific for compounds interacting with the human androgen receptor (AR), estrogen receptor α (ERα), progesterone receptor (PR), glucocorticoid receptor (GR), and peroxisome proliferator-activated receptor γ2 (PPARγ2). Indoor dust extracts were agonistic to ERα, GR, and PPARγ2 and antagonistic against AR, PR, GR, and PPARγ2. In comparison, a majority of FRs was agonistic to ERα and PPARγ2 only, and some FRs demonstrated receptor-specific antagonism against all tested nuclear receptors. Hierarchical clustering clearly indicated that agonism of ERα and antagonism of AR and PR were common, frequently detected end points for indoor dust and tested FRs. Given our previous results regarding the concentrations of FRs in indoor dust and in light of our current results, candidate contributors to these effects include not only internationally controlled brominated FRs but also alternatives such as some phosphorus-containing FRs. In the context of indoor pollution, high-frequency effects of FRs such as agonism of ERα and antagonism of AR and PR are candidate high-priority end points for further investigation.
The abnormal lipid metabolism in the liver that occurs after high caloric intake is the main cause of nonalcoholic fatty liver disease (NAFLD). Differences between samples from healthy livers and ...livers from individuals with NAFLD indicate that changes in liver function occur during disease progression. Here, we examined changes in protein expression in a fatty liver model in the early stages of obesity to identify potential alterations in function. The proteins expressed in the liver tissue of pre‐obese rats were separated via SDS/PAGE and stained with Coomassie brilliant blue‐G250. Peptide mass fingerprinting indicated an increase in the expression of carbonic anhydrase 3 (CA3) relative to controls. Western blotting analysis confirmed the increase in CA3 expression, even in an early fat‐accumulation state in which excessive weight gain had not yet occurred. In human hepatoma HepG2 cells, fat accumulation induced with oleic acid also resulted in increased CA3 expression. When the cells were in a state of fat accumulation, treating them with the CA3 inhibitors acetazolamide (ACTZ) or 6‐ethoxyzolamide (ETZ) suppressed fat accumulation, but only ETZ somewhat reduced the fat‐induced upregulation of CA3 expression. Expression of CA3 was therefore upregulated in response to the consumption of a high‐fat diet, even in the absence of an increase in body weight. The suppression of CA3 activity by ACTZ or ETZ reduced fat accumulation in hepatocytes, suggesting that CA3 is involved in the development of fatty liver.
Here, we have shown that carbonic anhydrase 3 (CA3) expression was enhanced as a result of high‐fat diet consumption, even in the absence of an excessive increase in body weight. In addition, suppression of CA3 expression and/or activity by 6‐ethoxyzolamide (ETZ) or acetazolamide (ACTZ) reduced fat accumulation in hepatocytes, suggesting that CA3 is involved in the development of fatty liver.
Phthalates are used in food packaging, and are transferred to foods as contaminants. In this study, we examined the hydrolytic metabolism of dimethyl phthalate (DMP), dibutyl phthalate (DBP) and ...di(2-ethylhexyl) phthalate (DEHP) by rat tissue microsomes. We found that carboxylesterase and lipase contribute differently to these activities. When DMP, DBP and DEHP were incubated with rat liver microsomes, DBP was most effectively hydrolyzed to the phthalate monoester, followed by DMP, and the activity toward DEHP was marginal. In contrast, small-intestinal microsomes exhibited relatively higher activity toward long-side-chain phthalates. Pancreatic microsomes showed high activity toward DEHP and DBP. Liver microsomal hydrolase activity toward DMP was markedly inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. The activity toward DBP and DEHP was partly inhibited by carboxylesterase inhibitor, and was partly solubilized with Triton X-100. Ces1e, Ces1d and Ces1f expressed in COS cells exhibited the highest hydrolase activity toward DBP, showing a similar pattern to that of liver microsomes. Ces1e showed activity towards DMP and DEHP. Pancreatic lipase also hydrolyzed DBP and DEHP. Thus, carboxylesterase and lipase contribute differently to phthalate hydrolysis: short-side-chain phthalates are mainly hydrolyzed by carboxylesterase and long-side-chain phthalates are mainly hydrolyzed by lipase.
•Tissue specificity of phthalate hydrolysis is related to side-chain length.•Rat liver microsomes hydrolyze dimethyl phthalate and dibutyl phthalate.•Small intestinal and pancreatic microsomes hydroyze di(2-ethylhexyl) phthalate.•Rat carboxylesterase isozymes hydrolyze dimethyl phthalate and dibutyl phthalate.•Pancreatic lipase hydrolyzes dibutyl phthalate and di(2-ethylhexyl) phthalate.
•Transesterification of parabens occurred when incubated with liver or small-intestinal microsomes, and alcohols.•Rat liver-type enzyme showed a preference for short-chain parabens, such as ...propylparaben.•Rat small-intestinal-type enzyme showed a preference for long-chain parabens, especially octylparaben.•Human liver and small-intestinal microsomes showed similar substrate specificities to those of rats.•Human CES1b and CES1c showed liver-type substrate specificity, and CES2 showed small-intestinal-type specificity.
Hydrolytic transformation of parabens (4-hydroxybenzoic acid esters; used as antibacterial agents) to 4-hydroxybenzoic acid and alcohols by tissue microsomes is well-known both in vitro and in vivo. Here, we investigated transesterification reactions of parabens catalyzed by rat and human microsomes, using a series of 12 parabens with C1–C12 alcohol side chains. Transesterification of parabens by rat liver and small-intestinal microsomes occurred in the presence of alcohols in the microsomal incubation mixture. Among the 12 parabens, propylparaben was most effectively transesterified by rat liver microsomes with methanol or ethanol, followed by butylparaben. Relatively low activity was observed with longer-side-chain parabens. In contrast, small-intestinal microsomes exhibited higher activity towards moderately long side-chain parabens, and showed the highest activity toward octylparaben. When parabens were incubated with liver or small-intestinal microsomes in the presence of C1–C12 alcohols, ethanol and decanol were most effectively transferred to parabens by rat liver microsomes and small-intestinal microsomes, respectively. Human liver and small-intestinal microsomes also exhibited significant transesterification activities with different substrate specificities, like rat microsomes. Carboxylesterase isoforms, CES1b and CES1c, and CES2, exhibited significant transesterification activity toward parabens, and showed similar substrate specificity to human liver and small-intestinal microsomes, respectively.
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