Effect of various chemicals on macromolecular binding during oxidative dealkylation of dimethylnitrosamine by hamster liver microsomes

Differential inhibitory effects of various chemicals on both formaldehyde formation and total macromolecular binding have been examined during oxidative demethylation of [¹⁴C]dimethylnitrosamine (DMN) by hamster liver microsomes. One group of chemicals such as diethyldithiocarbamate (DEDTC), aminoacetonitrile (AAN), 2-[(2,4-dichloro-6-phenyl) phenoxy]-ethylamine (DPEA), azide and ethanol inhibits both HCHO formation and total macromolecular binding. A second group of chemicals such as reduced glutathione, semicarbazide and N-(1-naphthyl)thiourea (NTU) inhibits macromolecular binding without appreciably affecting HCHO formation. Possible mechanisms of these differential inhibitory effects by these chemicals are discussed.     

Mutagenicity and antimutagenicity of hispidulin and hortensin, the flavonoids from Millingtonia hortensis L.

Studies of the mutagenicity and antimutagenicity of hispidulin and hortensin, the flavonoids from Millingtonia hortensis L. (Bignoniaceae), were performed using the liquid preincubation method of the Salmonella/microsome test. At the highest dose tested, 100 micrograms/plate, both compounds showed no mutagenicity and no cytotoxicity toward S. typhimurium strains TA98 and TA100 either in the presence or absence of S9 mix. However, these substances were antimutagens toward 2-aminoanthracene, aflatoxin B1 (in TA98), and dimethylnitrosamine (in TA100); but neither substance inhibited the direct mutagenic activity of 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide nor that of sodium azide in strains TA98 and TA100, respectively.     

Regulation of dimethylnitrosamine metabolism by androgenic hormones

Hormonal regulation of dimethylnitrosamine (DMN) metabolism by mouse kidney microsomes was investigated using an in vitro mutagenesis system that detects bioactive metabolites of this procarcinogen by measuring reverse mutation in Salmonella typhimurium his auxotrophs. Induction of microsomal DMN metabolizing enzymes was androgen-specific. Testosterone and medroxyprogesterone acetate were active inducers, d/1 norgestrel was less active, while epitestosterone, estradiol, and progesterone were ineffective. The response to testosterone and medroxyprogesterone acetate was time- and dose-dependent. Eleven strains of inbred mice were screened for their response to exogenous testosterone. DMN metabolism was stimulated in all mouse strains except for RF/J mice. Other androgenic end points were responsive in the latter strain, however. These observations suggest that induction of renal microsomal DMN metabolising enzymes is androgen-specific and probably mediated via the androgen receptor. The insensitivity of RF/J mice may be due to a mutation affecting a key step in the enzymatic activation of DMN.     

Metabolism-mediated cytotoxicity of chemical carcinogens and non-carcinogens

The results presented in this paper clearly demonstrate that a fibroblast suppression test system incorporating viable hepatocytes can be of value in studying the problems of metabolism-mediated cytotoxicity for those compounds and/or metabolites that are sufficiently stable to traverse cell membranes. The possibility that this test system can be used to distinguish carcinogenic and non-carcinogenic chemicals cannot be resolved at this time, however.     

Inhibition of CYP2E1 catalytic activity in vitro by S-adenosyl-L-methionine

The objective of this work was to evaluate the possible in vitro interactions of S-adenosyl-l-methionine (SAM) and its metabolites S-(5'-Adenosyl)-l-homocysteine (SAH), 5'-Deoxy-5'-(methylthio)adenosine (MTA) and methionine with cytochrome P450 enzymes, in particular CYP2E1. SAM (but not SAH, MTA or methionine) produced a type II binding spectrum with liver microsomal cytochrome P450 from rats treated with acetone or isoniazid to induce CYP2E1. Binding was less effective for control microsomes. SAM did not alter the carbon monoxide binding spectrum of P450, nor denature P450 to P420, nor inhibit the activity of NADPH-P450 reductase. However, SAM inhibited the catalytic activity of CYP2E1 with typical substrates such as p-nitrophenol, ethanol, and dimethylnitrosamine, with an IC(50) around 1.5-5mM. SAM was a non-competitive inhibitor of CYP2E1 catalytic activity and its inhibitory actions could not be mimicked by methionine, SAH or MTA. However, SAM did not inhibit the oxidation of ethanol to alpha-hydroxyethyl radical, an assay for hydroxyl radical generation. In microsomes engineered to express individual human P450s, SAM produced a type II binding spectrum with CYP2E1-, but not with CYP3A4-expressing microsomes, and SAM was a weaker inhibitor against the metabolism of a specific CYP3A4 substrate than a specific CYP2E1 substrate. SAM also inhibited CYP2E1 catalytic activity in intact HepG2 cells engineered to express CYP2E1. These results suggest that SAM interacts with cytochrome P450s, especially CYP2E1, and inhibits the catalytic activity of CYP2E1 in a reversible and non competitive manner. However, SAM is a weak inhibitor of CYP2E1. Since the K(i) for SAM inhibition of CYP2E1 activity is relatively high, inhibition of CYP2E1 activity is not likely to play a major role in the ability of SAM to protect against the hepatotoxicity produced by toxins requiring metabolic activation by CYP2E1 such as acetaminophen, ethanol, carbon tetrachloride, thioacetamide and carcinogens.     

Mutational response at the splenic T-Lymphocyte hprt locus in mice treated as neonates: Contrasting effects of the carcinogens N-ethyl-N-nitrosourea,dimethylnitrosamine, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

The newborn mouse tumorigenicity assay, which involves the treatment of animals during the first two weeks after birth and monitoring tumor induction after a year, has been suggested as a cost- and time-effective alternative to the conventional two-year rodent bioassay. In order to evaluate whether or not lymphocyte hprt mutant induction is an accurate predictor of carcinogenicity in the assay, we determined the frequencies of 6-thioguanine-resistant (TG^r) lymphocytes in the spleens of mice neonatally treated with the carcinogenic mutagens N-ethyl-N-nitrosourea (ENU), dimethylnitrosamine (DMN),and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Male C57BL/6 pups were injected on post-natal days 8 and 15, and the frequency of TG^r T-lymphocytes was measured in groups of three animals, sacrificed periodically up to 31 weeks post-treatment. Compared to background frequencies of 1.1–2.9 × 10⁻⁶, mutant frequencies (MFS) reached 155.1 × 10⁻⁶ following a cumulative dose of 49 mg ENU/kg body weight and 172.3 × 10⁻⁶ following a cumulative dose of 142 mg ENU/kg. These results show that TG^r lymphocyte mutations can be induced and measured in mice treated as neonates and that the induced MFs found for mice treated neonatally with ENU are comparable with frequencies reported for the treatment of adult animals with the same chemical. In contrast, treatment with the promutagenic and procarcinogenic compounds DMN (at a maximum concentration of 10.5 mg/kg) and PhIP (26.2 mg/kg) did not result in an increase in lymphocyte MF, suggesting that reactive metabolites of these compounds may not be reaching cells that are sensitive for mutation fixation. The results indicate that the lymphocyte hprt assay may fail to predict the carcinogenicity of some test chemicals in the neonatal mouse bioassay. Environ. Mol. Mutagen. 31:243–247, 1998 © 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Effect of precursors of endogenously synthesized carcinogen dimethylnitrosamine on activity of its demethylase in rat liver

Peroral administration of dimethylamine, sodium nitrite, and a combination of these two precursors of endogenously synthesized dimethylnitrosamine to rats increases the activity of the demethylase of this carcinogen in the liver microsomes. Under chronic experimental conditions the addition of dimethylamine to the rats' diet stimulates demethylase activity even if the diet contains casein, an inducer of this enzyme system. Actinomycin D, an inhibitor of protein synthesis, prevents the increase in demethylase activity in the microsomal fraction induced by dimethylamine.Laboratory of Dietary Carcinogenic Factors, Kiev Research Institute of Dietary Hygiene, Ministry of Health of the Ukrainian SSR. (Presented by Academician of the Academy of Medical Sciences of the USSR L. I. Medved'.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 12, pp. 717–719, December, 1977.     

木苏丸防治二甲基亚硝胺诱导的大鼠肝纤维化的实验研究

目的　观察木苏丸防治大鼠肝纤维化的疗效并探讨其作用机制。方法　采用 1%二甲基亚硝胺 (DMN) 10mg/ (kg·d)腹腔注射复制大鼠肝纤维化模型 ,木苏丸治疗组予木苏丸3.12 5 g/ (kg·d)灌胃 ,观察肝功能、血透明质酸 (HA)、超氧化物歧化酶 (SOD)、丙二醛 (MDA)含量变化及基质金属蛋白酶 - 2 (MMP - 2 )、金属蛋白酶组织抑制因子 - 1(TIMP - 1)、α -平滑肌肌动蛋白 (α -SMA)在肝组织中的表达 ;并观察肝脏显微结构改变。结果　木苏丸治疗组与模型对照组比较 ,碱性磷酸酶 (ALP)、MDA、HA明显下降 (P <0 .0 1和P <0 .0 5 ) ,血清白蛋白(ALB)、SOD明显增高 (P <0 .0 1) ,TIMP - 1表达下降 (P <0 .0 5 ) ,MMP - 2的表达无明显改变(P >0 .0 5 ) ;光镜下肝纤维化程度减轻。结论　木苏丸具有保肝、延缓肝纤维化形成的作用 ,其作用机制可能与其抗氧化及其下调TIMP - 1的表达有关     

Upregulation of TNF-α mRNA in hepatic fibrosis rats induced by dimethylnitrosamine

Objective：To observe the expression level of TNF-α mRNA in rats with hepatic fibrosis induced by dimethylnitrosamine （DMN） and to explore its relationship with collagen metabolism and its diagnostic value for hepatic fibrosis.Methods： Twenty-five male Wistar rats were randomly divided into normal control group （n=10） and model group （n=15）. Model rats were induced by DMN for 4 weeks and at final stage were executed. TNF-α mRNA were detected by RT-PCR and the inflammatory necrosis and collagen deposition in hepatic tissue were observed by HE stain and Sirius red stain. The liver functions were determined by automatic biochemical analytic device. The serum marks of liver fibrosis, such as HA, LN and Ⅳ-C were measured with ELISA and RIA. Results： In this study, the rat model of liver fibrosis induced by DMN was successfully constructed. RT-PCR reveals that TNF-α mRNA expression in control group is lower than that of model group. The liver functions of model group were impaired compared with those of the control group （P〈0.01）. Semi-quantitive analysis revealed that TNF-α/β-actin of normal rats was 0.39±0.12, while 0.93±0.05 of model rats. The concentration of HA （434.44±98.81 vs 252.9±26.59 ng/ml, P〈0.01）, LN （70.67±6.32 vs 37.90±5.97 ng/ml, P〈0.01） and Ⅳ-C （79.39±10.52 vs 21.40±4.17 ng/ml, P〈0.01） were significantly increased in the model group as well. Changes of the indexes were similar to the pathological damage of the liver. Conclusion： The results suggested that activation of TNF-α in liver tissues may be the common pathogenic mechanism of liver fibrosis. TNF-α may be a useful index for the diagnosis of hepatic fibrosis which worthies further investigation.     

Animal experiment and clinical study of effect of gamma-interferon on hepatic fibrosis

AIM To evaluate the antifibrotic effect ofdifferent doses of recombinant human Gamma-Interferon (IFN-γ) in two rat models of hepaticfibrosis, and to observe its effect on moderatechronic hepatitis B virus fibrosis.METHODS Hepatic fibrosis was successfullyinduced in 150 and 196 rats by subcutaneousinjection of carbon tetrachloride (CCl_4) andintraperitoneal injection of dimethylnitrosamine(DMN), respectively. Each of the two modelgroups was divided into: ① fibrotic modelgroup; ② colchicine treatment group (0.1 mg/kg/day, gastrogavage for 8 weeks); ③ high-dose IFN-γ group (15 MU/kg per day, i.m. for 8weeks); ④ medium-dose IFN-γ group (5 MU/kgdaily, i.m. for 8 weeks); and ⑤ low-dose IFN-γgroup (1.67 MU/kg daily, i.m. for 8 weeks).Another group of 10 rats without any treatmentwas used as normal controls. At the end of theexperiment, semi-quantitative histopathologicalscores of inflammation and fibrosis, liversmooth muscle actin (α-SMA) expression level,liver hydroxyl proline content and serumhyaluronic acid levels were compared. And 47medium chronic hepatitis B viral fibrosispatients were studied. They were given IFN-γtreatment, 100MU/day i,m. for the first threemonths and 100MU qod i.m. for the next sixmonths. Semi-quantitative pathological scoresof inflammation and fibrosis and serum hepaticfibrosis indices were compared within the 9months.RESULTS In animal experiment, thepathological fibrosis scores and liver hydroxylproline content were found to be significantly lower in rats treated with different doses of IFN-γ as compared with rats in fibrotic model groupinduced by either CCl_4 or DMN, in a dose-dependent manner. For CCl_4-induced model,pathological fibrosis scores in high, medium andlow doses IFN-γ groups were 5. 10±2.88, 7.70±3.53 and 8.00±3.30, respectively, but the scorewas 14.60±7.82 in fibrotic model group.Hydroxyl proline contents were 2.83±1.18, 3.59±1.22 and 4.80±1.62, in the three IFN-γgroups, and 10.01±3.23 in fibrotic model group.The difference was statistically significant(P<0.01). Similar results were found in DMN-induced model. Pathological fibrosis scoreswere 6.30±0.48, 8.10±2.72 and 8.30±2.58, inhigh, medium and low doses IFN-γ groups, and12.60±3.57 in fibrotic model group. Hydroxylproline contents were 2.72±0.58, 3.14±0.71and 3.62±1.02, in the three IFN-γ groups, and12.79±1.54 in fibrotic model group. Thedifference was statistically significant(P<0.01). Serum hepatic fibrosis indicesdecreased significantly in the 47 patients afterIFN-γ treatment (HA: 433.38±373.00 vs 281.57±220.48; LN: 161.22±41.02 vs 146.35±44.67;PCⅢ: 192.59±89.95 vs 156.98±49.22; C-Ⅳ:156.30±44.01 vs 139.14±34.47) and thedifferences between the four indices weresignificant (P<0.05). Thirty-three patientsreceived two liver biopsies, one before and oneafter IFN-γ treatment. In thirty of 33 patientsIFN-γ had better effects according to semi-quantitative pathological scores (8.40±5.83 vs5.30±4.05, P<0.05).CONCLUSION All the three doses of IFN-γ areeffective in treating rat liver fibrosis induced byeither CCl_4 or DMN, the higher the dose, thebetter the effect. And IFN-γ is effective forpatients with moderate chronic hepatitis B viralfibrosis.