Prenatal test cohort of a modified rat comparative thyroid assay adding brain thyroid hormone measurements and histology but lowering group size appears able to detect disruption by sodium phenobarbital.

The Comparative Thyroid Assay (CTA, USEPA) is a screening test for thyroid hormone (TH) disruption in peripheral blood of dams and offspring. Recently, we began investigating feasible improvements to the CTA by adding examination of offspring brain TH concentrations and brain histopathology. In addition, we hypothesize that the number of animals required could be reduced by 50 % while still maintaining sensitivity to characterize treatment related changes in THs. Previously, we showed that the prenatal test cohort of the modified CTA could detect 1000 ppm sodium phenobarbital (NaPB)-induced suppression of brain T3 (by 9 %) and T4 (by 33 %) with no significant changes in serum T3 and T4 (less than 8 %). In the current study we expanded the dose response in a prenatal test cohort. Pregnant SD rats (N = 10/group) were exposed to 0, 1000 or 1500 ppm NaPB in the diet from gestational days (GD) 6 to GD20. Serum THs concentrations in GD20 dams together with serum/brain THs concentrations and brain histopathology in the GD20 fetuses were examined. NaPB dose-dependently suppressed serum T3 (up to -26 %) and T4 (up to -44 %) in dams, with suppression of T3 in serum (up to -26 %) and brain (up to -18 %) and T4 in serum (up to -26 %) and brain (up to -29 %) of fetuses but without clear dose dependency. There were no remarkable findings that deviated significantly from controls in GD20 fetal brain by qualitative histopathology. Overall, the present study suggests that the prenatal test cohort of this modified CTA is able to detect the expected fetal TH disruptions by prenatal exposure to NaPB, while also reducing the number of animals used by 50 %, consistent with the results of our previous study. These findings add to the suggestion that lowering group sizes and adding endpoints may be a useful alternative to the original CTA design.

Chemical tolerance related to the ABC transporter gene and DNA methylation in cladocera (Daphnia magna).

We performed multigenerational tests to clarify the chemical tolerance mechanisms of a nontarget aquatic organism, Daphnia magna. We continuously exposed D. magna to a carbamate insecticide (pirimicarb) at lethal or sublethal concentrations (0, 3.8, 7.5, and 15 µg/L) for 15 generations (F0-F14). We then determined the 48 h-EC50 values and mRNA expression levels of acetylcholinesterase, glutathione S-transferase, and ATP (Adenosine triphosphate)-binding cassette transporter (ABCt) in neonates (<24 h old) from F0, F4, F9, and F14. To ascertain the effects of DNA methylation on pirimicarb sensitivity, we measured 5-methylcytosine levels (DNA methylation levels) in neonates of parents in the last generation (F14). In addition, we cultured groups exposed to 0 and 7.5 µg/L (the latter of which acquired chemical tolerance to pirimicarb) with or without 5-azacytidine (de-methylating agent) and determined methylation levels and 48 h-EC50 values in neonates (<24 h old) from the treated parents. The EC50 values (30.3-31.6 µg/L) in F14 of the 7.5 and 15 µg/L groups were approximately two times higher than that in the control (16.0 µg/L). A linear mixed model analysis showed that EC50 and ABCt mRNA levels were significantly increased with generational alterations; further analysis showed that the ABCt mRNA level was positively related to the EC50 . Therefore, ABCt may be associated with altered pirimicarb sensitivity. In addition, the EC50 value and DNA methylation levels in pirimicarb-tolerant clones decreased after exposure to 5-azacytidine, suggesting that DNA methylation contributes to chemical tolerance. These findings improved our knowledge regarding the acquisition of chemical tolerance in aquatic organisms.

Feasibility study for a downsized comparative thyroid assay with measurement of brain thyroid hormones and histopathology in rats: Case study with 6-propylthiouracil and sodium phenobarbital at high dose.

Concern has been raised that thyroid hormone disruptors (THDs) may potentially interfere with the developing brain, but effects of mild suppression of maternal THs by environmental contaminants on neonatal brain development are not fully understood. The comparative thyroid assay (CTA) is a screening test for offspring THDs, but it requires several animals and is criticized that reliance on serum THs alone as predictive markers of brain malfunction is inadequate. To verify feasibility of the downsized CTA but additional examination of brain THs levels and histopathology, we commenced internal-validation studies. This paper presents the data of the study where 6-propylthiouracil (6-PTU, 10 ppm) and sodium phenobarbital (NaPB, 1000 ppm) were dosed by feeding from gestational days (GD)6-20, and from GD6 to lactation day 21. The modified CTA detected 6-PTU-induced severe (>70%) suppression of serum THs in dams, with >50% suppressed serum/brain TH levels in offspring and brain heterotopia in postnatal day 21 pups. The modified CTA also detected NaPB-induced mild (<35%) suppression of serum THs in dams, with mild (<35%) reduction of serum/brain TH levels in fetuses but not in pups. These findings suggest that the modified CTA may have a potential as a screening test for offspring THDs.

[Separate Analysis of Loquat Fruit Pulp, Peel, and Pits to Calculate Pesticide Residue Levels in the Whole Commodity].

To evaluate the effects of handling "not detectable" residues (ND: <0.01 mg/kg) in the pulp and detectable residues in the pits on the calculation of pesticide residue in the whole fruit, residue levels in the pulp, peel, and pits of loquat fruits were separately analyzed. Following conventional Japanese agricultural practices, 16 pesticides were sprayed at the maximum application rates in three test fields. All target pesticides were detected at quantifiable levels in the peel (n=144). In contrast, the percentages of detected pesticides in the pulp and pits were 42% (n=61) and 36% (n=52), respectively. Most pesticide residues were present in the peel. For comparison, the pesticide residue levels in the whole fruits were determined based on three indices: the highest estimate (H), calculated using the measured residue levels in the pits and by replacing the ND residues in the pulp as the limit of quantification (LOQ) values; conventional estimate (C), calculated by neglecting all residues in the pits (0 mg/kg) and replacing the ND residues in the pulp as LOQ values; and the lowest estimate (L), calculated by neglecting all residues in the pits and the ND residues in the pulp (0 mg/kg). The L/C and H/C ratios ranged from 74% (L/C) to 106% (H/C). In seven of eighty-three cases with less than 90% difference, residue levels in the whole loquat fruits were low (≤0.06 mg/kg), with the actual range being equal to or below the minimum unit of 0.01. In comparison of three field datasets, the range of residue levels was estimated to be 2.77 mg/kg. Based on the results of separate analysis, handling of ND residues in the pulp and detectable residues in the pits did not significantly affect the calculated pesticide residue levels in the whole loquat fruits.

Possible enzymatic mechanism underlying chemical tolerance and characteristics of tolerant population in Scapholeberis kingi.

To determine the potential effects of pesticides on aquatic organisms inhabiting a realistic environment, we explored the characteristics and mechanisms of chemical tolerance in Scapholeberis kingi(Cladocera). We established a chemical-tolerant population via continuous exposure to pirimicarb, an acetylcholinesterase (AChE) inhibitor, and examined the effects of pirimicarb concentration on the intrinsic growth rates (r) of tolerant cladocerans. We also explored the association between r and feeding rate and tested the involvement of antioxidant enzymes [peroxidase (PO) and superoxide dismutase] and AChE in pirimicarb sensitivity. S. kingi was continuously exposed to lethal and sublethal pirimicarb concentrations (0, 2.5, 5, and 10 µg/L) for 15 generations, and changes (half maximal effective concentration at 48 h, 48 h-EC50) in chemical sensitivity were investigated. In the F14 generation, the sensitivity of the 10 µg/L group was three times lower than that of the control group, suggesting the acquisition of chemical tolerance. Moreover, r was significantly and negatively correlated with 48 h-EC50, suggesting a fitness cost for tolerance. Surprisingly, there was no significant correlation between r and feeding rate. There was a weak but significant positive correlation between each enzyme activity and the 48 h-EC50 value (p < 0.05). Thus, oxidative stress regulation and enhanced AChE may be involved in the acquisition of chemical tolerance in cladocerans. These findings will help elucidate the characteristics and mechanisms of chemical tolerance in aquatic organisms.

Generational sensitivity alteration in Chironomus yoshimatsui to carbamate and pharmaceutical chemicals and the effect on Catalase, CYP450, and hemoglobin gene transcription.

To ascertain the tolerance mechanisms of aquatic organisms to artificial chemicals, intergenerational sensitivity changes of Chironomus yoshimatsui to a carbamate pesticide (pirimicarb) and pharmaceutical chemical (diazepam) were investigated. The larvae (<48-h-old) in each generation were exposed to both chemicals for 48 h and then the surviving chironomids were cultured until the fifth generation (F0-F4) without chemical addition. The 48-h 50% effective concentration (EC50) value of chironomids was determined for each generation. In the pirimicarb treatment group, the EC50 values significantly increased in F3 and F4, and those in the diazepam treatment group slightly increased. Catalase, Cytochrome P450 and hemoglobin (Hb) mRNA levels were monitored to see whether these were related to the trans-generational sensitivity. Although the generalized linear model results showed that the sensitivity to diazepam was slightly increased in the diazepam treatment, we could not find any mRNA levels related to sensitivity alteration. In contrast, the model approach showed that the chironomids exposed to pirimicarb trans-generationally became tolerant with increasing Hb mRNA levels. Therefore, they might decrease their chemical stress by modifying Hb gene transcription.

Rapid adaptation of Chironomus yoshimatsui to acetylcholinesterase inhibitors (pyraclofos and pirimicarb) in a multi-generation study.

We evaluated the real effects of pollutants through a multi-generation study. We tested whether short-term exposure (48 h) of successive (first and second) generations of Chironomus yoshimatsui neonates (<24-h-old) to two acetylcholinesterase inhibitor insecticides, pyraclofos, and pirimicarb, would change insecticide sensitivity and life-cycle parameters over four generations. Additionally, we tested whether acetylcholinesterase (AChE) activity levels would be associated with this sensitivity change. Sensitivities (48 h EC50 value, using immobility as the endpoint) in chironomids (<24-h-old) and insect life-cycle parameters (the number of larvae per egg mass and adult size) were investigated. Parental chironomids produced larvae that were less sensitive than those in the control group following the two 48 h pirimicarb exposure events, whereas exposure to pyraclofos did not affect sensitivity. The AChE activity in larvae with low sensitivity to pirimicarb was significantly higher than that in the control. Thus, increased AChE activity might be associated with low sensitivity. The life-cycle parameters in chironomids recovered from the effects of pyraclofos and pirimicarb suggested they could adapt to the insecticides by changing biomass allocation. Our study suggested potential chemical risks of insecticide stress and how aquatic organisms adapt to it.

Toxicity and Carcinogenicity of Dichlorodiphenyltrichloroethane (DDT).

Dichlorodiphenyltrichloroethane (DDT) is still used in certain areas of tropics and subtropics to control malaria and other insect-transmitted diseases. DDT and its metabolites have been extensively studied for their toxicity and carcinogenicity in animals and humans and shown to have an endocrine disrupting potential affecting reproductive system although the effects may vary among animal species in correlation with exposure levels. Epidemiologic studies revealed either positive or negative associations between exposure to DDT and tumor development, but there has been no clear evidence that DDT causes cancer in humans. In experimental animals, tumor induction by DDT has been shown in the liver, lung, and adrenals. The mechanisms of hepatic tumor development by DDT have been studied in rats and mice. DDT is known as a non-genotoxic hepatocarcinogen and has been shown to induce microsomal enzymes through activation of constitutive androstane receptor (CAR) and to inhibit gap junctional intercellular communication (GJIC) in the rodent liver. The results from our previously conducted 4-week and 2-year feeding studies of p,p'-DDT in F344 rats indicate that DDT may induce hepatocellular eosinophilic foci as a result of oxidative DNA damage and leads them to hepatic neoplasia in combination with its mitogenic activity and inhibitory effect on GJIC. Oxidative stress could be a key factor in hepatocarcinogenesis by DDT.

A further study on the reliability of toxicokinetic parameters for predicting hepatotoxicity in rats receiving a 28-day repeated administration of DDT.

We previously demonstrated a good correlation between the increased relative liver weight caused by DDT and the area under the concentration-time curve (AUC) of DDT or the total DDT (T-DDT) in plasma and liver of rats in a 7-day repeated dose study at 1000 ppm. To confirm the reliability of AUC for predicting toxic responses at different dose levels, we conducted a further 28-day repeated dose study of p,p'-DDT in male F344 rats at dietary levels of 50, 160, and 500 ppm. Concentrations of DDT and its metabolites in plasma, brain, liver, and fat for each dose group were measured at various time intervals during the study. The concentrations of DDT and T-DDT in plasma and liver were compared with their AUCs in relation to hepatotoxic responses including increased relative liver weight, microsomal enzyme induction (CYP2B1), and inhibition of gap junctional intercellular communication. The coefficient (R(2)) values of each toxic response in correlation with AUCs were generally much higher than those with concentrations at any dose levels, although the R(2) values vary considerably among toxic parameters. These results have confirmed that the AUC of DDT or T-DDT in plasma or liver is a reliable marker for predicting hepatotoxicity caused by DDT in repeated dose studies.

Mechanisms of promotion and progression of preneoplastic lesions in hepatocarcinogenesis by DDT in F344 rats.

Time-related changes in potential factors involved in hepatocarcinogenesis by DDT were investigated in a 4-week and a 2-year feeding studies of p,p'-DDT with F344 rats. In the 4-week study with males at doses of 50, 160, and 500 ppm, cell proliferation and gap junctional intercellular communication (GJIC) were examined after 1, 2, 3, 7, 14, and 28 days. Cell proliferation was enhanced within 3 days at any dose level, but returned to normal after 7 days, whereas GJIC was inhibited throughout the study. In the 2-year study with both sexes at doses of 5, 50, and 500 ppm, cell proliferation, GJIC, enzyme induction, and oxidative stress were investigated after 26, 52, 78, and 104 weeks. Males and females showed an inhibition of GJIC and increases in P450 isozymes (CYP2B1 and CYP3A2) in a dose-dependent manner at all time points, but no significant change in cell proliferation. Lipid peroxide for males at 50 and 500 ppm and 8-hydroxydeoxyguanosine for both sexes at 500 ppm were elevated throughout the study. Histologically, eosinophilic foci and hepatocellular adenomas increased in males at 50 ppm and both sexes at 500 ppm. Hepatocellular carcinomas also developed in males at 500 ppm. These results indicate that DDT may induce eosinophilic foci as a result of oxidative DNA damage and leads them to neoplasms in combination with its mitogenic activity and inhibitory effect on GJIC. Oxidative stress could be a key factor in hepatocarcinogenesis by DDT.

A comparative study on the reliablility of toxicokinetic parameters for predicting hepatotoxicity of DDT in rats receiving a single or repeated administration.

A comparative study on the reliability of toxicokinetic parameters for predicting hepatotoxicity was conducted in male F344 rats receiving a single (106 mg/kg by gavage) or 7-day repeated (1000 ppm in feed, 97 mg/kg/day) administration of p,p'-DDT. DDT was selected as the test substance because it is known as a hepatotoxic agent and its metabolic pathway is well documented. Concentrations of DDT and its metabolites (DDE and DDD) in the plasma, brain, and liver were measured at various time intervals during the study and the results were compared with the area under the concentration-time curve (AUC) in relation to hepatotoxic response. Increases in the absolute and relative (ratio to body weight) liver weights were observed as a typical toxic response after a single or repeated exposure to DDT. The coefficient (R2) of correlation between the increases in the relative liver weight and the concentrations or AUC of DDT and its metabolites in the plasma and liver was estimated. The values of R2 between the relative liver weight and AUC of DDT or the total DDT (T-DDT) in the plasma and liver were found to be more consistent and higher than those with their concentrations in the repeated dose study. In addition, the R2 values in correlation with their AUCs after a single exposure were lower than those in the repeated dose study. These results indicate that the AUC of DDT or T-DDT in the plasma and liver would be more reliable than their concentrations for predicting hepatotoxicity caused by DDT, especially in the repeated dose study.