Disruption of spindle checkpoint function in rats following 28 days of repeated administration of renal carcinogens.

We previously reported that 28-day exposure to hepatocarcinogens that facilitate cell proliferation specifically alters the expression of G1/S checkpoint-related genes and proteins, induces aberrant early expression of ubiquitin D (UBD) at the G2 phase, and increases apoptosis in the rat liver, indicating G1/S and spindle checkpoint dysfunction. The present study aimed to determine the time of onset of carcinogen-specific cell-cycle disruption after repeated administration of renal carcinogens for up to 28 days. Rats were orally administered the renal carcinogens nitrofurantoin (NFT), 1-amino-2,4-dibromoantraquinone (ADAQ), and 1,2,3-trichloropropane (TCP) or the non-carcinogenic renal toxicants 1-chloro-2-propanol, triamterene, and carboxin for 3, 7 or 28 days. Both immunohistochemical single-molecule analysis and real-time RT-PCR analysis revealed that carcinogen-specific expression changes were not observed after 28 days of administration. However, the renal carcinogens ADAQ and TCP specifically reduced the number of cells expressing phosphorylated-histone H3 at Ser10 in both UBD(+) cells and proliferating cells, suggestive of insufficient UBD expression at the M phase and early transition of proliferating cells from the M phase, without increasing apoptosis, after 28 days of administration. In contrast, NFT, which has marginal carcinogenic potential, did not induce such cellular responses. These results suggest that it may take 28 days to induce spindle checkpoint dysfunction by renal carcinogens; however, induction of apoptosis may not be essential. Thus, induction of spindle checkpoint dysfunction may be dependent on carcinogenic potential of carcinogen examined, and marginal carcinogens may not exert sufficient responses even after 28 days of administration.

Successful validation of genomic biomarkers for human immunotoxicity in Jurkat T cells in vitro.

Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6 h to subcytotoxic doses of nine immunotoxicants, five non-immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)-PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non-immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo-e-pyrene). Of the four potential (non-)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non-immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3-dichloro-propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e. chlorpyrifos, aldicarb, benzo-e-pyrene and anti-CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT-PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non-immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity.

HOCl-modified phosphatidylcholines induce apoptosis and redox imbalance in HUVEC-ST cells.

Electrophilic attack of hypochlorous acid on unsaturated bonds of fatty acyl chains is known to result mostly in chlorinated products that show cytotoxicity to some cell lines and were found in biological systems exposed to HOCl. This study aimed to investigate more deeply the products and the mechanism underlying cytotoxicity of phospholipid-HOCl oxidation products, synthesized by the reaction of HOCl with 1-stearoyl-2-oleoyl-, 1-stearoyl-2-linoleoyl-, and 1-stearoyl-2-arachidonyl-phosphatidylcholine. Phospholipid chlorohydrins were found to be the most abundant among obtained products. HOCl-modified lipids were cytotoxic towards HUVEC-ST (endothelial cells), leading to a decrease of mitochondrial potential and an increase in the number of apoptotic cells. These effects were accompanied by an increase of the level of active caspase-3 and caspase-7, while the caspase-3/-7 inhibitor Ac-DEVD-CHO dramatically decreased the number of apoptotic cells. Phospholipid-HOCl oxidation products were shown to affect cell proliferation by a concentration-dependent cell cycle arrest in the G0/G1 phase and activating redox sensitive p38 kinase. The redox imbalance observed in HUVEC-ST cells exposed to modified phosphatidylcholines was accompanied by an increase in ROS level, and a decrease in glutathione content and antioxidant capacity of cell extracts.

[Toxicity of dichloropropanols].

A rare outbreak of acute hepatic damage in workers exposed to dichloropropanols was reported in 1992. As there are no detailed reports of dichloropropanols (DCPs) toxicity and its mechanism, we reviewed the toxicity of dichloropropanols using our results. 1) A marked elevation of serum AST and ALT with massive necrosis of the liver was noted in the 1/2 x, the 1 x and 2 x LD50 (0.149 mg/kg) of 1, 3-dichloro-2-propanol(DC 2 P). Hepatic malondialdehyde level was significantly increased, and associated with a decrease in liver glutathione S-transferase activity and reduced glutathione content. It is suggested that the free radical is associated with DCPs. 2) A reduction of leukocytes, platelets and fibrinogen, and prolonged prothrombin time were observed in the 1 x LD50 of DC 2 P. 3) In the CA1 area of the hippocampus, inhibition of population spikes was reduced by the 1 x LD50 of DC 2 P. This research was completed with the assistance of several other papers concerning dichloropropanols toxicity.

Mutagenic potential of bisphenol A diglycidyl ether (BADGE) and its hydrolysis-derived products in the Ames Salmonella assay.

The mutagenicity of bisphenol A diglycidyl ether (BADGE), its first and second hydrolysis products (the diol epoxide and bis-diol of BADGE, respectively) and the bis-chlorohydrin of BADGE were investigated using the Ames Salmonella assay with strains TA98, TA100, TA1535 and TA1537. The assays were performed in the absence and presence of various concentrations of rat liver S9 fraction. The results obtained confirm the mutagenic power of BADGE in strains TA100 and TA1535 and show a positive response to the diol epoxide of BADGE in these strains, although the latter compound was approximately 10 times less potent than the former. A lack of mutagenic activity of the bis-diol of BADGE and the chlorohydrin under study is also shown. These findings suggest that BADGE and, to a much lesser extent, the diol epoxide of BADGE may constitute a genotoxic hazard, but not the bis-diol or bis-chlorohydrin of BADGE.

Fatty acid chlorohydrins and bromohydrins are cytotoxic to human endothelial cells.

Reaction of unsaturated lipids with the hypohalous acids (hypochlorous acid and hypobromous acid) results in the addition of the halide (X) across double bonds to form halohydrins (-CH2CH(OH)CH(X)CH2-). These modified lipids could be potentially destabilising to cell membranes due to their increased polarity. We have investigated the effect of pre-formed halohydrins on human umbilical vein endothelial cells (HUVEC) by incubating cultured cells with oleic acid micelles containing chlorohydrins or bromohydrins. Cell detachment and necrotic death were observed with increasing doses of halohydrins, whereas the cells were unaffected by equivalent doses of oleic acid. Bromohydrins caused more lysis than did chlorohydrins at equivalent doses. Complete lysis was seen with 200 microM fatty acid/chlorohydrin micelles and with 50 microM fatty acid/bromohydrin micelles. Chlorohydrin uptake was much less than the oleic acid control whereas bromohydrins were incorporated into the endothelial cells similarly to oleic acid. This difference or the bulkier nature of the bromohydrins could account for their increased toxicity. This study has demonstrated the potential toxicity of the halohydrins, and implications for their formation in inflammation are discussed.

Effect of cyanamide on toxicity and glutathione depletion in rat hepatocyte cultures: differences between two dichloropropanol isomers.

The effect of aldehyde dehydrogenase inhibition by cyanamide pre-treatment in vitro on dichloropropanol-dependent toxicity and glutathione depletion was investigated in 24 h rat hepatocyte cultures. Cyanamide pre-treatment had no effect on nitrophenol hydroxylase, 7-methoxy-, 7-ethoxy- or 7-benzyloxyresorufin O-dealkylase activities in 24 h cultures from untreated rats, and had no effect on intracellular glutathione content in cultures from untreated rats, or in cultures from isoniazid-treated rats in which cytochrome P4502E1 (CYP2E1) is increased. In cultures from untreated animals the primary alcohol, 2,3-dichloropropanol, was not toxic and did not significantly deplete glutathione. Cyanamide pre-treatment however, potentiated both toxicity and glutathione depletion. Induction of CYP2E1 also potentiated the toxicity of 2,3-dichloropropanol, and in these cultures cyanamide pre-treatment significantly increased both toxicity and glutathione depletion. Cyanamide did not alter the toxicity or glutathione depletion due to the secondary alcohol, 1,3-dichloropropanol, irrespective of CYP2E1 induction. These results indicate that the primary alcohol isomer is metabolised to an aldehyde intermediate which depletes glutathione. Under basal conditions this metabolite appears to be effectively detoxified, but increased CYP2E1 activity and/or decreased aldehyde dehydrogenase activity promotes accumulation of metabolite, and therefore increases glutathione depletion and toxicity.

Involvement of cytochrome P4502E1 in the toxicity of dichloropropanol to rat hepatocyte cultures.

Hepatocytes were isolated and cultured from untreated rats and rats treated with isoniazid to induce cytochrome P4502E1. Isoniazid selectively increased p-nitrophenol hydroxylase activity in 2-h cultures, and increased the toxicity of both 1,3- and 2,3-dichloropropanol. Isoniazid also increased the rate and extent of glutathione depletion by the dichloropropanols. There was no effect of isoniazid on the toxicity of 1,3-dichloroacetone, precocene II or allyl alcohol. In addition, diethyldithiocarbamate selectively inhibited p-nitrophenol hydroxylase in 2-h cultures from untreated and isoniazid-treated rats, as well as abolishing toxicity of the dichloropropanols. In 24-h cultures from isoniazid-treated rats diethyldithiocarbamate inhibited high affinity MCOD activity by 55% and there was also a small but significant inhibition of precocene II toxicity. These results indicate that isoniazid-inducible P4502E1 can mediate the toxicity of dichloropropanol.

Mutagenic and carcinogenic risk of oxygen containing chlorinated C-3 hydrocarbons: putative secondary products of C-3 chlorohydrocarbons and chlorination of water.

Oxygen containing C-3 chlorohydrocarbons are secondary products of C-3 chlorohydrocarbons formed during oxidation at air, in the metabolism of pesticides and by chlorination of drinking water. These compounds are mutagenic, genotoxic and carcinogenic. 2-Chloroacroleins are extremely strong mutagens and genotoxins and form 1,N2-cyclic deoxyguanosine adducts. The role of such adducts in mutagenicity and carcinogenicity is discussed.

Liver injury induced by dichloropropanols--changes in the time course on hematological and blood chemical examinations.

The toxicity of dichloropropanols (DCPs) was investigated by hematological and blood chemical examination. Solutions of two isomers of DCPs, 1,3-dichloro-2-propanol (DC2P) and 2,3-dichloro-1-propanol (DC1P) were dissolved in saline at a concentration of 100 mg/ml and 0.1 ml of each was subcutaneously injected into male Wistar rats weighing about 200 g. Acute changes on transaminases and number of platelets were determined in the time course. 6 hours later, transaminases showed significant increases while the number of platelets significantly decreased in the DC2P-treated group. In the half the of DC2P-treated group, transaminases had increased furthermore at 24 hours, while those in the rest were recovered to the control level. No changes were observed in the DC1P-treated group. These results indicate that there is a prominent hepatotoxicity in DC2P, with the individual diversities to some extent and the hepatic toxicity differs considerably between DC2P and DC1P. Therefore, the monitoring of the working environment and biological monitoring of DCPs should be mandatory, in the workplace where DCPs, especially DC2P, are utilized.

Chemical mutagenesis testing in Drosophila. IX. Results of 50 coded compounds tested for the National Toxicology Program.

Fifty chemicals were tested for mutagenic activity in post-meiotic and meiotic germ cells of male Drosophila melanogaster using the sex-linked recessive lethal (SLRL) assay. As in the previous studies in this series, feeding was chosen as the first route of administration. If the compound failed to induce mutations by this route, injection exposure was used. One gaseous chemical (1,3-butadiene) was tested only by inhalation. Those chemicals that were mutagenic in the sex-linked recessive lethal assay were further tested for the ability to induce reciprocal translocations. Eleven of the 50 chemicals tested were mutagenic in the SLRL assay. These included bis(2-chloroethyl) ether, 1,4-butanediol diglycidyl ether, 1-chloro-2-propanol, dimethyl methylphosphonate, dimethyl morpholinophosphoramidate, dimethyloldihydroxyethylene urea, 2,2-dimethyl vinyl chloride, hexamethylphosphoramide, isatin-5-sulfonic acid (Na salt), isopropyl glycidyl ether, and urethane. Five of these, including 1,4-butanediol diglycidyl ether, 2,2-dimethyl vinyl chloride, hexamethylphosphoramide, isopropyl glycidyl ether, and urethane, also induced reciprocal translocations.

[Toxicity of dichloropropanols--changes in hematological findings and serum chemistry].

We investigated the toxicity of dichloropropanols (DCPs) in hematological findings and serum chemistry. The solutions of two isomers of DCPs, 1,3-dichloro-2-propanol (DC2P) and 2,3-dichloro-1-propanol (DC1P) were dissolved in saline at the concentration of 100 mg/ml, and 0.1 ml of each solution was subcutaneously injected into male Wistar rats weighing about 200 g. At 6 hours after the injections, in the DC2P group, the number of white blood cells and platelets showed a significant decrease. Transaminases, alkaline phosphatase and lactate dehydrogenase were greatly elevated. Blood urea nitrogen and creatinine also showed a significant increase. There were no changes in the measurements in the DC1P group. These results indicate that there is a prominent hepatotoxicity in DC2P, and that there is a considerable difference in the toxicity present in DC2P and DC1P. Furthermore, in the workplace where DCPs, especially DC2P, is used, the monitoring of the working environment and biological monitoring should be mandatory.

Effects of epichlorohydrin on male and female reproduction in Long-Evans rats.

Male and female Long-Evans rats were treated with epichlorohydrin (ECH) by oral gavage (males: 12.5, 25, and 50 mg/kg/day; females: 25, 50, and 100 mg/kg/day) for 21 and 14 days, respectively, prior to mating trials with untreated animals. Treated females were further dosed until delivery. Fertility was assayed in the high-dose males only and was found to be totally impaired. No measured parameters of female reproduction were changed relative to controls. Treated males showed normal copulatory behavior. Sperm morphology and percentage motile sperm were not statistically different from control values in both ejaculated and cauda epididymal samples from ECH-treated animals. The number of sperm in ejaculates was normal while cauda epididymal sperm count was slightly decreased in males at the 50 mg ECH/kg dose level. Mean curvilinear velocity, straight-line velocity, and amplitude of lateral head displacement of cauda epididymal sperm were significantly reduced by ECH at 12.5 mg/kg/day and above. Sperm track linearity was also reduced, but only at 50 mg/kg/day. Beat/cross frequency of sperm was significantly increased at 12.5 mg/kg/day and above. All of the above sperm motion parameters showed dose-dependent trends. These effects are consistent with the spermatozoal metabolic lesions reported for alpha-chlorohydrin, a metabolite of ECH.

2,2,2-Trifluoroethanol intestinal and bone marrow toxicity: the role of its metabolism to 2,2,2-trifluoroacetaldehyde and trifluoroacetic acid.

2,2,2-Trifluoroethanol (TFE) produces bone marrow and small intestine toxicity resulting in leukopenia, loss of intestinal dry weight, and consequent lethal septicemia in male Wistar rats. Its metabolic pathway, based on serum and small intestine time courses of substrate and metabolites, was determined to be TFE in equilibrium 2,2,2-trifluoroacetaldehyde (TFAld)----trifluoroacetic acid (TFAA). Administered TFE and TFAld were not toxic per se, since their toxicity and metabolism were inhibited by pyrazole. TFE and TFAld were equipotent at equimolar doses thus precluding the oxidative reaction, TFE to TFAld, from being the toxic step. Since equimolar TFAA exhibited no toxic effects, an oxidative intermediate on the pathway from TFAld to TFAA, most likely F3C-C+(OH)2, must thus be the toxic moiety. The intermediate TFAld is stable in serum, as determined by a novel assay developed for its analysis in biological systems, and can be transported to the target tissues, bone marrow, and small intestine, after formation probably in the liver. On the basis of the more rapid metabolism of TFE to higher levels of TFAld in the small intestine and bone marrow than in the serum, the closer correspondence of bone marrow and small intestine metabolite ratios than serum ratios at high and low doses of TFE to the corresponding ratios of toxicity, and the decreased toxicity of TFAld when administered ig versus ip, the formation of the toxic metabolic intermediate of TFE probably occurs in the target tissues.