Antioxidant and Pulmonary Protective Potential of Fraxinus xanthoxyloides Bark Extract against CCl4 -Induced Toxicity in Rats.

Fraxinus xanthoxyloides is a perennial shrub belonging to family Oleaceae, traditionally used for malaria, jaundice, pneumonia, inflammation, and rheumatism. Our study is aimed to assess the total phenolics (TPC), flavonoids (TFC), terpenoids contents (TTC) and antioxidant profiling of F. xanthoxyloides methanol bark extract (FXBM) and its fractions, hexane, chloroform, ethyl acetate and aqueous, along with high-performance liquid chromatography with diode-array detection (HPLC-DAD). Further, the antioxidant and pulmonary protective potential was explored against carbon tetrachloride (CCl4 )-induced CCl4-induced pulmonary tissue damage in rats. The highest TPC, TFC and TTC were found in FXBM (133.29±4.19 mg/g), ethyl acetate fraction (279.55±10.35 mg/g), and chloroform fraction (0.79±0.06 mg/g), respectively. The most potent antioxidant capacity was depicted by FXBM (29.21±2.40 µg/mg) and ethyl acetate fraction (91.16±5.51 µg/mg). The HPLC-DAD analysis revealed the predominance of gallic, chlorogenic, vanillic and ferulic acid in FXBM. The administration of CCl4 induced oxidative stress, suppressed antioxidant enzymes' activities including catalase, peroxidase, superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, and glutathione reductase. Further, it increased thiobarbituric acid reactive substances (TBARS) and H2 O2 levels, induced DNA injuries and reduced the total protein and glutathione content in lung tissues. The treatment of rats with FXBM restored these biochemical parameters to the normal level. Moreover, the histopathological studies of lung tissues demonstrated that FXBM protected rats' lung tissues from oxidative damage restoring normal lung functions. Thus, F. xanthoxyloides bark extract is recommended as adjuvant therapy as protective agent for patients with lung disorders.

Environmental risk appraisement of disinfection by-products (DBPs) in plant model system: Allium cepa.

The organic toxicants formed in chlorinated water cause potential harm to human beings, and it is extensively concentrated all over the world. Various disinfection by-products (DBPs) occur in chlorinated water are genotoxic and carcinogenic. The toxicity is major concern for chlorinated DBPs which has been present more in potable water. The purpose of the work was to evaluate genotoxic properties of DBPs in Allium cepa as a plant model system. The chromosomal aberration and DNA laddering assays were performed to examine the genotoxic effect of trichloroacetic acid (TCAA), trichloromethane (TCM), and tribromomethane (TBM) in a plant system with distinct concentrations, using ethyl methanesulfonate (EMS) as positive control and tap water as negative control. In Allium cepa root growth inhibition test, the inhibition was concentration dependent, and EC50 values for trichloroacetic acid (TCAA), trichloromethane (TCM), and tribromomethane (TBM) were 100 mg/L, 160 mg/L, and 120 mg/L respectively. In the chromosome aberration assay, root tip cells were investigated after 120 h exposure. The bridge formation, sticky chromosomes, vagrant chromosomes, fragmented chromosome, c-anaphase, and multipolarity chromosomal aberrations were seen in anaphase-telophase cells. It was noticed that with enhanced concentrations of DBPs, the total chromosomal aberrations were more frequent. The DNA damage was analyzed in roots of Allium cepa exposed with DBPs (TCAA, TCM, TBM) by DNA laddering. The biochemical assays such as lipid peroxidation, H2O2 content, ascorbate peroxidase, guaiacol peroxidase, and catalase were concentration dependent. The DNA interaction studies were performed to examine binding mode of TCAA, TCM, and TBM with DNAs. The DNA interaction was evaluated by spectrophotometric and spectrofluorometric studies which revealed that TCAA, TCM, and TBM might interact with Calf thymus DNA (CT- DNA) by non-traditional intercalation manner.

No significant impact of Foxf1 siRNA treatment in acute and chronic CCl4 liver injury.

Chronic liver injury of any etiology is the main trigger of fibrogenic responses and thought to be mediated by hepatic stellate cells. Herein, activating transcription factors like forkhead box f1 are described to stimulate pro-fibrogenic genes in hepatic stellate cells. By using a liver-specific siRNA delivery system (DBTC), we evaluated whether forkhead box f1 siRNA treatment exhibit beneficial effects in murine models of acute and chronic CCl4-induced liver injury. Systemic administration of DBTC-forkhead box f1 siRNA in mice was only sufficient to silence forkhead box f1 in acute CCl4 model, but was not able to attenuate liver injury as measured by liver enzymes and necrotic liver cell area. Therapeutic treatment of mice with DBTC-forkhead box f1 siRNA upon chronic CCl4 exposition failed to inhibit forkhead box f1 expression and hence lacked to diminish hepatic stellate cells activation or fibrosis development. As a conclusion, DBTC-forkhead box f1 siRNA reduced forkhead box f1 expression in a model of acute but not chronic toxic liver injury and showed no positive effects in either of these mice models. Impact statement As liver fibrosis is a worldwide health problem, antifibrotic therapeutic strategies are urgently needed. Therefore, further developments of new technologies including validation in different experimental models of liver disease are essential. Since activation of hepatic stellate cells is a key event upon liver injury, the activating transcription factor forkhead box f1 (Foxf1) represents a potential target gene. Previously, we evaluated Foxf1 silencing by a liver-specific siRNA delivery system (DBTC), exerting beneficial effects in cholestasis. The present study was designed to confirm the therapeutic potential of Foxf1 siRNA in models of acute and chronic CCl4-induced liver injury. DBTC-Foxf1 siRNA was only sufficient to silence Foxf1 in acute CCl4 model and did not ameliorate liver injury or fibrogenesis. This underlines the significance of the experimental model used. Each model displays specific characteristics in the pathogenic nature, time course and severity of fibrosis and the optimal time point for starting a therapy.

Cytotoxic and phytotoxic actions of Heliotropium strigosum.

This study describes the cytotoxic and phytotoxic activities of the crude extract of Heliotropium strigosum and its resultant fractions. In brine shrimp toxicology assays, profound cytotoxicity was displayed by ethyl acetate (LD50 8.3 µg/ml) and chloroform (LD50 8.8 µg/ml) fractions, followed by relatively weak crude methanolic extract of H. strigosum (LD50 909 µg/ml) and n-hexane fraction (LD50 1000 µg/ml). In case of phytotoxicity activity against Lemna acquinoctialis, highest phytotoxic effect was showed by ethyl acetate fraction (LD50 91.0 µg/ml), while chloroform fraction, plant crude extract and n-hexane, respectively, caused 50%, 30.76 ± 1.1% and 30.7 ± 1.1% inhibitory action at maximum concentration used, that is, 1000 µg/ml. These data indicates that H. strigosum exhibits cytotoxic and phytotoxic potential, which explore its use as anticancer and herbicidal medicine. The ethyl acetate and chloroform fractions were more potent for the evaluated toxicity effects, thus recommended for isolation and identification of the active compounds.

Evaluation of the genotoxic potential of Austroplenckia populnea (Reiss) Lundell chloroform fraction from barkwood extract in rodent cells in vivo.

The genotoxic effect of the Austroplenckia populnea chloroform fraction from barkwood extract was tested in vivo on peripheral blood cells of Swiss mice with the comet assay (SCGE), and the clastogenic effect was investigated on peripheral blood cells of Swiss mice and bone marrow cells of Wistar rats, with the micronucleus and chromosome aberrations tests. The animals were treated by gavage with 3 concentrations of the extract: 300, 600 and 900 mg.kg-1. Peripheral blood cells of Swiss mice were collected 4 and 24 hours after the treatment to the SCGE assay and 48 and 72 hours to the micronucleus test. Bone marrow cells of Wistar rats were collected 24 hours after the treatment to the micronucleus and chromosome aberration tests. The results showed that the A. populnea chloroform fraction induced an increase in the average number of DNA damage in peripheral blood cells at the three concentrations tested, but this increase was not statistically significant. In the micronucleus and chromosome aberrations test, no significant increase was observed in the mean number of micronucleated polychromatic erythrocytes (MNPCE) of Swiss mice or MNPCE or chromosome aberrations for the rat bone marrow cells, for any of the tested doses. Our findings enable us to conclude that by the comet assay, A. populnea chloroform fraction from barkwood extract showed no genotoxic effects, and by the micronucleus and chromosome aberration tests, the extract fraction showed no clastogenic/aneugenic effects on the rodent cells tested.

Evaluation of the genotoxic potential of Austroplenckia populnea (Reiss) Lundell chloroform fraction from barkwood extract in rodent cells in vivo / Avaliação do potencial genotóxico da fração clorofórmica de cascas do caule de Austroplenckia populnea em células de roedores in vivo

The genotoxic effect of the Austroplenckia populnea chloroform fraction from barkwood extract was tested in vivo on peripheral blood cells of Swiss mice with the comet assay (SCGE), and the clastogenic effect was investigated on peripheral blood cells of Swiss mice and bone marrow cells of Wistar rats, with the micronucleus and chromosome aberrations tests. The animals were treated by gavage with 3 concentrations of the extract: 300, 600 and 900 mg.kg-1. Peripheral blood cells of Swiss mice were collected 4 and 24 hours after the treatment to the SCGE assay and 48 and 72 hours to the micronucleus test. Bone marrow cells of Wistar rats were collected 24 hours after the treatment to the micronucleus and chromosome aberration tests. The results showed that the A. populnea chloroform fraction induced an increase in the average number of DNA damage in peripheral blood cells at the three concentrations tested, but this increase was not statistically significant. In the micronucleus and chromosome aberrations test, no significant increase was observed in the mean number of micronucleated polychromatic erythrocytes (MNPCE) of Swiss mice or MNPCE or chromosome aberrations for the rat bone marrow cells, for any of the tested doses. Our findings enable us to conclude that by the comet assay, A. populnea chloroform fraction from barkwood extract showed no genotoxic effects, and by the micronucleus and chromosome aberration tests, the extract fraction showed no clastogenic/aneugenic effects on the rodent cells tested.

O possível efeito genotóxico da fração clorofórmica de cascas de caule de Austroplenckia populnea foi testado in vivo em células do sangue periférico de camundongos Suíços pelo ensaio cometa, e o seu possível efeito clastogênico foi investigado em células de sangue periférico de camundongos Suíços e células da medula óssea de ratos Wistar, respectivamente pelos testes do micronúcleo e de aberrações cromossômicas. Os animais foram tratados por via oral com três concentrações do extrato: 300, 600 e 900 mg.kg-1 de peso corpóreo. Células do sangue periférico dos camundongos foram coletadas 4 e 24 horas após o tratamento para a realização do ensaio cometa, e 48 e 72 horas para o teste do micronúcleo. Células da medula óssea dos ratos Wistar foram coletadas 24 horas após o tratamento para os testes do micronúcleo e de aberrações cromossômicas. Os resultados mostraram que a fração clorofórmica do extrato de A. populnea induziu um pequeno aumento no número médio de danos ao DNA nas células sanguíneas nas três concentrações testadas, mas tal aumento não foi estatisticamente significativo. Nos testes do micronúcleo e de aberrações cromossômicas não houve um aumento significativo no número médio de eritrócitos policromáticos micronucleados (EPM) dos camundongos, bem como nos EPM e aberrações cromossômicas nas células da medula óssea dos ratos, em nenhuma das doses testadas. Nossos resultados nos permitem concluir que, pelo ensaio cometa, a fração clorofórmica de cascas do caule de A. populnea não apresentou efeito genotóxico, e, pelos testes do micronúcleo e de aberrações cromossômicas, o extrato não mostrou efeitos clastogênicos e/ou aneugênicos nas células analisadas dos roedores.

In vitro evaluation of macrophage viability after incubation in orange oil, eucalyptol, and chloroform.

OBJECTIVE: The aim of this study was to evaluate the cytotoxicity of orange oil, eucalyptol, and chloroform in a cell culture assay by using peritoneal macrophages from Swiss mice. STUDY DESIGN: Control (Dulbecco's modified Eagle's medium [DMEM] plus 1.25% ethyl alcohol) and experimental (orange oil, eucalyptol, and chloroform) groups were studied. Solvents used were tested at 0.025% and 0.050% concentrations in DMEM plus 1.25% ethyl alcohol. RESULTS: Orange oil, eucalyptol, and chloroform were all cytotoxic in comparison to the control group (P < .001). Orange oil showed the least cytotoxicity (P < .001). No significant differences were observed regarding cell viability when comparing the eucalyptol and chloroform groups (P < .05). There were significant differences in the cytotoxicity of eucalyptol and chloroform with an increase in concentration (P < .0001). Nevertheless, this difference was not significant in the orange oil group (P < .05). CONCLUSION: Orange oil was less cytotoxic than eucalyptol and chloroform.

The phytotoxic effect of C(1)/C(2)-halocarbons and trichloroacetic acid on the steppe plant Artemisia lerchiana.

Artemisia lerchiana is a wormwood species of the Central Asian steppe regions, where it completely cover whole areas. For the first time it was possible to show through field experiments that C(1)/C(2) halocarbons (VCHCs), such as chloroform (CHL), tetrachloroethene (PER) and hexachloroethane (HEX), can be taken up by test plants of the species A. lerchiana via the soil/root pathway and metabolised inter alia into trichloroacetic acid (TCA) under semi-aride conditions. At the same time, chlorophyll a fluorescence measurements carried out on the test plants revealed a phytotoxic influence on plant vitality (max. decline in vitality of 52% with application of CHL) and less efficient energy flows in the photosynthesis mechanism of the A. lerchiana test plants. The authors examine possible links between the simultaneous appearance of VCHCs and additional drought stress in the acceleration of desertification processes.

Comparative study of different silymarin formulations: formulation, characterisation and in vitro/in vivo evaluation.

The aim of the present study was to study the synergistic hepatoprotective effect of silymarin with phospholipids when it is encaged in microspheres so as to passively target it to liver and to compare these silymarin formulations with silymarin solution. Various silymarin loaded lipid emulsions were formulated which include formulation A prepared with soyabean oil as an internal oily phase, soya lecithin as surfactant and tween 80 as cosurfactant; formulation B which was same as formulation A but was filtered through 0.45 micro membrane filter and finally steam sterilized for intravenous administration; formulation C containing soyabean oil as an internal oily phase, soya lecithin as surfactant, tween 80 and propylene glycol as cosurfactant/ cosolvent. These formulations were compared for their release profile with silymarin solution in propylene glycol, i.e. formulation D. In vivo evaluation was carried out using three models i.e. phenobarbitone induced sleep time in mice, biochemical estimation of SGOT and SGPT enzyme levels and histopathological examination of rat livers. Results revealed that there was significant reduction in sleep time in the mice treated with silymarin loaded lipid microspheres (both p.o. as well as i.v.) when compared with control and even with plain lipid microspheres and silymarin solution and significant reduction in enzyme levels in silymarin lipid microspheres treated group when compared with control, plain lipid microspheres as well as silymarin solution treated group. Histopathological studies also supported the results obtained from the other two models. A positive outcome of these studies gave an insight that if silymarin is coupled with phospholipid in such microparticulate delivery systems, hepatoprotective effect of drug molecules can be pronounced further by self targeting nature and synergistic action.

Effects of betaine supplementation on hepatic metabolism of sulfur-containing amino acids in mice.

BACKGROUND/AIMS: We previously reported that acute betaine treatment induced significant changes in the hepatic glutathione and cysteine levels in mice and rats. The present study was aimed to determine the effects of dietary betaine on the metabolism of sulfur-containing amino acids. METHODS/RESULTS: Male mice were supplemented with betaine (1%) in drinking water for up to 3 weeks. Changes in hepatic levels of major sulfur amino acid metabolites and products were stabilized after 2 weeks of betaine supplementation. Betaine intake increased methionine, S-adenosylmethionine, and S-adenosylhomocysteine levels significantly, but homocysteine and cystathionine were reduced. Methionine adenosyltransferase activity was elevated to three-fold of control. Cysteine catabolism to taurine was inhibited as evidenced by a decrease in cysteine dioxygenase activity and taurine levels in liver and plasma. Despite the significant changes in the transsulfuration reactions, neither hepatic cysteine nor glutathione was altered. Betaine supplementation decreased the hepatotoxicity induced by chloroform (0.5 ml/kg, ip) significantly. CONCLUSIONS: Betaine supplementation enhances recycling of homocysteine for the generation of methionine and S-adenosylmethionine while reducing its utilization for the synthesis of cystathionine and cysteine. However, the hepatic levels of cysteine or glutathione are not affected, most probably due to the depression of taurine generation from cysteine.

Analysis of preneoplastic and neoplastic renal lesions in Tsc2 mutant Long-Evans (Eker) rats following exposure to a mixture of drinking water disinfection by-products.

Disinfection of surface water for human consumption results in the generation of a complex mixture of chemicals in potable water. Cancer risk assessment methodology assumes additivity of carcinogenic effects in the regulation of mixtures. A rodent model of hereditary renal cancer was used to investigate the carcinogenic response to a mixture of drinking water disinfection by-products (DBPs). Rats carrying a mutation in the Tsc2 tumor suppressor gene (Eker rats) readily develop renal preneoplastic and neoplastic lesions, and are highly susceptible to the effects of renal carcinogens. Male and female Eker rats were exposed via drinking water to individual or a mixture of DBPs for 4 or 10 months. Potassium bromate, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), chloroform, and bromodichloromethane were administered at low concentrations of 0.02, 0.005, 0.4 and 0.07 g/l, respectively, and high concentrations of 0.4, 0.07, 1.8 and 0.7 g/l, respectively. Low and high dose mixture solutions were comprised of all four chemicals at either low concentrations or high concentrations, respectively, Following necropsy, each kidney was examined microscopically for preneoplastic lesions (atypical tubules and hyperplasias) and tumors. While some of the mixture responses observed in male rats did fall within the range expected for an additive response, especially at the high dose, predominantly antagonistic effects on renal lesions were observed in response to the low dose mixture in male rats and the high dose mixture in female rats. These data suggest that current default risk assessments assuming additivity may overstate the cancer risk associated with exposure to mixtures of DBPs at low concentrations.

Effect of dosing vehicle on the hepatotoxicity of CCl4 and nephrotoxicity of CHCl3 in rats.

There are conflicting results in the literature concerning the effect of gavage vehicle, corn oil (CO) versus aqueous suspension, on the toxicity of haloalkanes. The purpose of our study was to assess the influence of oral dosing vehicle on the acute hepatotoxicity of CCl4 and nephrotoxicity of CHCl3. Male Sprague-Dawley rats, fed ad libitum, were treated (po) with single doses of CCl4 or CHCl3 using corn oil (CO), or an aqueous preparation (5%) of Emulphor (EL620) or Tween-85 (Tw-85) as vehicle (10 ml/kg). Rats were killed 48 h after treatment. Blood was collected for plasma alanine aminotransferase (ALT) determination and renal cortical slices were prepared for p-aminohippuric acid (PAH) incorporation. The comparison, between gavage vehicles, of the slopes and ED50 of the dose-response curves, although not significantly different, indicated clear trends for enhanced potency with CO for CHCl3 nephrotoxicity but not for CCl4 hepatotoxicity. However, ALT values, a measure of the severity of effect for CCl4, also indicated that CO, when compared to EL620 and Tw-85, tended to enhance CCl4 hepatotoxicity at low toxicity incidence. Furthermore, CO clearly enhanced the severity of effect for CHCl3 nephrotoxicity, as measured by the slice-to-medium PAH ratios, at high dosage. The greater severity of the lesion produced by exposure to these chemicals, when administered in CO, is consistent with the trends observed for their potency (dose-response curves). Our results agree with an increased toxicity of haloalkanes by the gavage vehicle CO reported in the literature. Thus, CO should be considered a potential confounder in hepato- and nephrotoxicity assays.

Chloroform in drinking water prevents hepatic cell proliferation induced by chloroform administered by gavage in corn oil to mice.

Chloroform administered by gavage in corn oil, but not when administrated in drinking water, has been shown to induce liver cancer in female B6C3F1 mice and to enhance cell proliferation. Since humans are exposed to chloroform in their drinking water, we evaluated whether exposure by this route would interact with the activity of chloroform when administered by gavage in corn oil. Female B6C3F1 mice were exposed to chloroform in drinking water for 33 days at 0, 300, or 1800 ppm (Experiment 1) or for 31 days at 0, 120, 240, or 480 ppm (Experiment 2) and for 3 days prior to termination also received a daily dose of 263 mg/kg chloroform administered by gavage in corn oil. Exposure to chloroform in drinking water reduced both the hepatotoxicity and the enhanced cell proliferation (bromodeoxyuridine-labeling index and mitotic index) elicited in response to chloroform administered by gavage in corn oil. Hence, chloroform administered in drinking water reduced the activity of chloroform administered by gavage in corn oil, suggesting that it would also reduce the hepatocarcinogenic activity of chloroform administered by gavage.

Induced regenerative cell proliferation in livers and kidneys of male F-344 rats given chloroform in corn oil by gavage or ad libitum in drinking water.

These studies were designed to establish the dose response relationships for the induction of cytolethality and regenerative cell proliferation in the liver and kidneys of male F-344 rats given chloroform by gavage or in drinking water. Rats were administered oral doses of 0, 10, 34, 90 or 180 mg/kg/day chloroform dissolved in corn oil by gavage for 4 days or for 5 days/week for 3 weeks. A second group of rats was given chloroform ad libitum in the drinking water at concentrations of 0, 60, 200, 400, 900 or 1800 ppm for 4 days or 3 weeks. Bromodeoxyuridine (BrdU) was administered via an implanted osmotic pump 3.5 days prior to necropsy to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the labelling index (LI) evaluated as the percentage of S-phase cells. Rats treated with 90 or 180 mg/kg/day by gavage for 4 days had mild to moderate degeneration of renal proximal tubules and centrilobular hepatocytes. These alterations were absent or slight after 3 weeks of treatment. LI were increased in the kidney cortex only in the rats treated with 180 mg/kg/day for 4 days. A dose-dependent increase in LI was seen in rat liver after 4 days of treatment with 90 and 180 mg/kg/day by gavage, but the LI remained elevated after 3 weeks of treatment only at the 180 mg/kg/day dose. When chloroform was administered in the drinking water, no microscopic alterations were seen in the kidneys after 4 days of treatment. As a general observation, rats treated for 3 weeks with 200 ppm chloroform and greater had slightly increased numbers of focal areas of regenerating renal proximal tubular epithelium and cell proliferation than were noted in the controls, but no clear dose response relationship was evident. However, the overall renal LI was not increased at any dose or time point. Similarly, only mild hepatocyte vacuolation was observed in rats given 1800 ppm chloroform in the water for 3 weeks with no increase in the hepatic LI at any time point, even though the rats were consuming chloroform at a rate of 106 mg/kg/day at the 1800 ppm drinking water concentration. These data indicate more severe hepatic and renal toxicity when chloroform is administered by gavage than in the drinking water and a different pattern of regenerative proliferation in the kidney.(ABSTRACT TRUNCATED AT 400 WORDS)

Route of administration determines whether chloroform enhances or inhibits cell proliferation in the liver of B6C3F1 mice.

Chloroform administered by gavage has been shown to induce liver cancer in B6C3F1 mice; however, when administered in the drinking water, chloroform inhibited liver cancer in mice. The effect of chloroform administered by these two routes upon cell proliferation in mouse liver was determined. Female B6C3F1 mice were divided into five treatment groups: (1) chloroform (263 mg/kg body wt) by gavage in corn oil, (2) 1,800 ppm chloroform in drinking water, (3) 1,800 ppm chloroform in drinking water plus 10 ml/kg body wt corn oil by gavage, (4) 10 ml/kg body wt corn oil by gavage, and (5) untreated controls. Five days prior to termination, the mice were implanted subcutaneously with a 7-day osmotic minipump containing 30 mg/ml bromodeoxyuridine. Mice were terminated after 5, 12, 33, and 159 days of exposure. Chloroform administered by gavage in corn oil increased cell proliferation at all terminations, while, when administered in drinking water, cell proliferation was inhibited on Days 5 and 12. At 33 and 159 days, chloroform administered in the drinking water did not affect cell proliferation, even though the dose received by the animals was comparable to that given in corn oil by gavage. Therefore, cell proliferation was enhanced only by chloroform administered in corn oil by gavage, which corresponds to the hepatocarcinogenicity of chloroform administered by this route.

Effects of chloroform and bromodichloromethane on DNA synthesis in male F344 rat kidney.

We have been investigating the actions of chloroform (CHCl3) and bromodichloromethane (BDCM) in rat kidney after different routes of exposure. Male F344 rats were exposed by gavage with corn oil or water as the diluting vehicle. All experiments lasted 30 days with gavage exposures 5 days per week for 4 weeks (20 doses). All animals were injected IP with bromodeoxyuridine (BrdU) 3 times over a 6-day period at 50 mg/kg/injection. Kidney tissue was fixed and slides were stained with hematoxylin and eosin for routine viewing and by the PAP (peroxidase-antiperoxidase) technique using anti-BrdU to label cells in DNA synthesis. There were no significant changes in gross parameters evaluated between the control rats and the rats exposed to CHCl3 or BDCM. Rats exposed via corn oil gavage to CHCl3 displayed a segment-specific epithelial cell necrosis (6/6 high dose, 2/6 low dose). The lesions were primarily localized to the second segment of the proximal tubule, although some spread to cells in the first segment was occasionally observed. No histologic lesions were observed in the kidneys of rats exposed to BDCM. Preliminary results indicate a significant increase in DNA synthesis in the CHCl3-treated rats and a slight increase in DNA synthesis in BDCM-treated rats with corn oil as the diluent. The increase in BrdU labeling was primarily in cells of the S2 segment of the proximal tubule and interstitial cells of CHCl3-exposed animals and in cells of the S3 segment of BDCM-exposed animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of the hepatotoxicity of chloroform in B6C3F1 mice by corn oil: implications for chloroform carcinogenesis.

A recent study of the ability of chloroform in drinking water to produce cancer reported that male Osborne-Mendel rats developed renal tumors, but that female B6C3F1 mice failed to develop hepatocellular carcinomas. The results obtained in the male Osborne-Mendel rats were comparable to those observed in an earlier study sponsored by the National Cancer Institute (NCI). On the other hand, the lack of an increased incidence of hepatocellular carcinomas in female B6C3F1 mice was in sharp contrast to previously reported results. The doses of chloroform used were comparable to that which produced an 85% incidence in the NCI study. We have investigated the extent to which the vehicle might be responsible for the different results in these two studies by examining the differential effects of chloroform when it was administered by gavage using corn oil versus a 2% Emulphor suspension as the vehicle. Male and female B6C3F1 mice were administered chloroform at 60, 130, and 270 mg/kg per day for 90 days. At sacrifice, body and organ weights were measured, and blood was recovered to perform the following serum chemistry measurements (in order of priority): glutamate oxalacetate transaminase (SGOT), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), and triglyceride (TG) levels. The liver was sectioned for histopathological examination. Chloroform increased SGOT levels significantly only when administered in corn oil at a dose of 270 mg/kg in both male and female mice. It had no effect on LDH activity. There was a small increase in BUN when chloroform was administered in corn oil, but not when administered in 2% Emulphor. When administered in corn oil, chloroform significantly decreased serum TG levels but was without effect on this parameter when administered in 2% Emulphor. Chloroform decreased body weight and increased liver weight with both vehicles, but the effects were significantly greater when it was administered in corn oil. Mice administered chloroform in corn oil displayed a significant degree of diffuse parenchymal degeneration (5 of 10 males and 1 of 10 females) and mild to moderate early cirrhosis (5 of 10 males and 9 of 10 females); significant pathological lesions were not observed in the animals administered corn oil without chloroform nor in mice receiving chloroform in 2% Emulphor. These data indicate that administration of chloroform by corn oil gavage results in more marked hepatotoxic effects than observed when it is provided in an aqueous suspension.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolism of toxic substances in isolated hepatocytes.

Chloroform and selenite toxicity have been studied in isolated hepatocytes. Two different toxic mechanisms, which lead to cellular lysis after distinct lag periods, are compared. Chloroform toxicity can be divided into 2 phases, a first phase characterized by chloroform metabolism and a second phase characterized by lipid peroxidation. GSH depletion during the first phase is claimed to be a prerequisite for lipid peroxidation in the second phase. Selenite metabolism leads to cofactor depletion as well. Selenite reduction via a GSH reductase dependent pathway exhausts the cells of NADPH and this effect can be related to cellular lysis.