An important role for cytosol in the microsomal metabolism of N-nitrosodimethylamine to a mutagen: evidence for two different mutagenic metabolites

Microsomal-mediated mutagenesis induced by N-nitrosodimethylamine (NDMA) in Salmonella TA100 at neutral pH was only slightly affected by cytosol and was similar in its threshold type dose-response curve to mutagenesis induced by direct-acting N-nitroso-N-methyl compounds. However, mutagenesis in strain TA104 was greatly enhanced by cytosol and this mutagenesis did not exhibit a threshold. In the presence of microsomes alone NDMA was more potent in TA100 than TA104, but in the presence of microsomes plus cytosol (S-9 fraction) this order was reversed at the doses tested. A possible explanation for these results is that NDMA is metabolized by microsomes to a mutagen (presumably methyldiazonium ion; MDI) that is more potent in TA100 than in TA104, but in the presence of S-9 fraction a fraction of the NDMA is metabolized by a pathway leading to a different mutagen with a different specificity. The ratio of metabolism via these pathways appears to be dependent on pH.     

二甲基亚硝胺与大鼠肝组织DNA形成加合物的定量研究

作者应用免疫组化技术结合图象分析技术,对二甲基亚硝胺(N-nitrosodimethylamine,NDMA)诱导大鼠肝脏DNAO~6-甲基鸟嘌呤(Omethylguanine,OmG)作了定性和定量研究。分别给大鼠一次腹腔注射NDMA1,2,3,5,10,30,50mg/kg,5小时后大鼠肝脏产生O(?)mG显示明显的剂量反应关系和细胞特异性。肝组织中细胞产生O(?)mG的量与NDMA的剂量间呈幂函数关系,二者呈正相关;产生O(?)mG的细胞数与NDMA的剂量间呈指数函数关系,二者呈正相关。肝细胞、肝小叶中央静脉和肝窦内皮细胞显示O(?)mG阳性。     

Steepness of the radiation dose-response curve for dose-per-fraction escalation keeping the number of fractions fixed

Clinically, there is growing interest in strategies for intensifying radiation therapy by escalating the dose per fraction. This paper considers the steepness of the dose-response curve in this case. The steepness of a radiation dose-response curve is most conveniently quantified by the normalized dose-response gradient, γ. Under the assumption of a linear-quadratic dose-effect model, a simple analytical relationship is derived between the γ-value for a dose-response curve generated by varying the total dose while keeping the number of fractions constant, i.e. escalating the dose per fraction, and the γ-value for a dose-response curve generated by varying the total dose while keeping the dose per fraction constant. This formulation is compared with clinical dose-response data from the literature and shown to be in good agreement with the observations. Some implications of this formulation for non-uniform dose distributions delivered using 3D conformal radiotherapy or intensity modulated radiotherapy (IMRT) are briefly discussed.     

Factors regulating activation and DNA alkylation by 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone and nitrosodimethylamine in rat lung and isolated lung cells, and the relationship to carcinogenicity

The molecular dosimetry for O6-methylguanine (O6MG) formation in DNA from rat lung and pulmonary cells was compared following treatment for 4 days with equimolar doses of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent pulmonary carcinogen or nitrosodimethylamine (NDMA), a weak carcinogen in rat lung. The dose response for O6MG formation from NNK was biphasic; the O6MG to dose ratio, an index of alkylation efficiency, increased dramatically as the dose of carcinogen was decreased. In contrast, the dose-response curve for methylation by NDMA appeared opposite of that for NNK with alkylation efficiency increasing as a function of dose. These results suggested that high and low Km pathways exist for the activation of NNK, whereas only high Km pathways may be involved in NDMA activation. Furthermore, DNA methylation by NNK was cell selective with the highest levels in the Clara cell, whereas methylation by NDMA was not. DNA methylation in the Clara cell was 50-fold greater by NNK than by NDMA at equimolar doses (0.005 mmol/kg). Thus, differences in O6MG formation, specifically the presence of a high affinity pathway in the Clara cell for activation of NNK, may explain why following low dose exposure, NNK is a potent pulmonary carcinogen while NDMA is not. Different cytochrome P-450 isozymes also appear to be involved in the activation of NNK and NDMA. Inhibition of in vitro methylation (with calf thymus DNA and lung microsomes) by antibodies to cytochrome P-450 isozymes provided evidence that a homolog of rabbit cytochrome P-450(2) (cytochrome P-450b) may be important in the activation of NNK in rat lung, whereas cytochrome P-450(5) may activate NDMA. A 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible cytochrome P-450 isozyme (P-450c) may also be involved in the activation of NNK but not NDMA. Treatment with TCDD increased both NNK activation by pulmonary microsomes and the formation of O6MG in Clara cells and type II cells incubated in vitro with NNK. alpha-Naphthoflavone (alpha-NF), a specific inhibitor of cytochrome P-450c reversed the increase in methylation by TCDD-induced microsomes but did not inhibit in vitro activation of NNK using microsomes from untreated rats. However, NNK mediated O6MG formation in Clara cells, but not in type II cells incubated with alpha-NF, was decreased by 21%. These data indicate that both cytochrome P-450b and P-450c are probably involved in the activation of NNK in Clara cells from untreated rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lack of potential of low dose N-nitrosodimethylamine to induce preneoplastic lesions, glutathione S-transferase placental form-positive foci, in rat liver

Induction of liver lesions in male F344 rats by the genotoxic and carcinogenic N-nitrosodimethylamine (NDMA) was studied at a wide range of dose levels, i.e. from 0.001 to 10 ppm, in drinking water for 16 weeks. Dose related and statistically significant increase of glutathione S-transferase placental form-positive foci, endpoint markers for hepatocarcinogenesis in rats, at 1 and 10 ppm dose groups was obtained, but no increment in foci could be detected with the lower doses (0.001, 0.01, and 0.1 ppm). This finding of a no-observed effect level supports our hypothesis that a threshold, at least in practical terms, exists in carcinogenesis proposed on the basis of extensive wide range dose-dependence studies of other genotoxic carcinogens.     

Threshold dose dependence in phenobarbital promotion of rat hepatocarcinogenesis initiated by diethylnitrosamine.

The dose-response of phenobarbital (PB) promotion of hepatocarcinogenesis in rats was investigated. Male F344 rats were given 1, 4, 16, 75, 300 or 1200 p.p.m. PB solutions given ad libitum as their drinking water for 39 weeks following initiation with a single i.p. injection of diethylnitrosamine (DEN) (100 mg/kg). At week 40, the incidence of hepatic tumors was increased clearly in the DEN + PB groups given 300 p.p.m. PB or above, as compared to that in the group given DEN only. Linear dose-response curves for numbers and sizes of enzyme-altered hepatic foci (gamma-glutamyl-transpeptidase or placental glutathione S-transferase positive foci) were obtained in the dose range 16-1200 p.p.m. PB. The minimum promoting dose level of PB for enzyme-altered foci, estimated from dose-response curves by the Logit model, was calculated to be 15-23 p.p.m. Thus while dose dependence was demonstrated over a large range, a threshold was evident at low doses.     

二甲基亚硝胺诱导大鼠DNA O^6—甲基鸟嘌呤的免疫组化研究

本文应用双PAP法对二甲基亚硝胺(N-nitrosodjmethylamine,NDMA)诱导大鼠DNAO~6—甲基鸟嘌呤(O~5—methylguanine,O~6—mG)进行了研究。分别给大鼠一次腹腔注射NDMA 1,2,3,5,10,30,50mg/kg,5小时后大鼠产生O~6—mG具有明显的剂量反应关系以及器官和细胞特异性。随着NDMA剂量的增加,产生O~6—mG的器官种类和细胞类型增加,阳性细胞形成O~6—mG的量也增加。小剂量(1—5mg/kg)NDMA仅见肝组织的细胞形成O~6—mG,NDMA剂量达50mg/kg时,肝、肾、肺、气管,食管和鼻咽组织的细胞均检出O~6—mG。     

Existence of no-observed effect levels for 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline on hepatic preneoplastic lesion development in BN rats

There is increasing evidence that dose-response curve of genotoxic carcinogen is nonlinear and a practical threshold dose exists. However, little is known about differences in the dose-response relationship of genotoxic carcinogen among different strain rats. Herein, we showed that low doses of genotoxic carcinogen 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx) had no effects on induction of liver glutathione S-transferase placental form (GST-P)-positive foci in both BN and F344 rats, and therefore demonstrated the existence of no-observed effect level for hepatocarcinogenicity of this genotoxic carcinogen irrespective of strains. These findings further support our notion that a practical threshold dose for MeIQx hepatocarcinogenicity exists in rats.     

N-NITROSODIMETHYLAMINE: HAZARD CHARACTERIZATION AND EXPOSURE–RESPONSE ANALYSIS

N-Nitrosodimethylamine (NDMA) has been assessed as a Priority Substance under the Canadian Environmental Protection Act. Based upon laboratory studies in which tumours have been induced in all species examined at relatively low doses, NDMA is clearly carcinogenic, with a very strong likelihood that the mode of action for the induction of tumours involves direct interaction with genetic material. Qualitatively, the metabolism of NDMA appears to be similar in humans and animals; as a result, it is considered highly likely that NDMA is carcinogenic to humans. A Tumorigenic Dose₀₅ (TD₀₅) of 34 μg/kg body weight per day has been derived, based upon the benchmark dose associated with a 5% increase in the development of hepatic biliary cystadenomas in female rats in an oral carcinogenicity bioassay.     

Heterologous expression of rat P450 2E1 in a mammalian cell line: in situ metabolism and cytotoxicity of N-nitrosodimethylamine

GM0637, a human fibroblast cell line, was transfected with pCMV2E1, an expression vector containing the full length cDNA for rat cytochrome P450 2E1 (P450 2E1), and with pCMVneo, which contained vector alone, and the selected clones were designated GM2E1 and GMneo, respectively. Western blot analysis showed that GM2E1, but not GMneo, expressed a protein that reacted with anti-human P450 2E1 antibody. The 7- ethoxycoumarin O-deethylase,p-nitrophenol hydroxylase, and N- nitrosodimethylamine (NDMA) demethylase activities of the P450 in these cells were measured in monolayer cell cultures without preparing microsomes. Exposure of the GM2E1 cells to NDMA for 4 days caused severe decreases in cell viability, as determined by crystal violet uptake, and showed a sigmoidal dose-response curve with a median lethal dose of 17 microM. In contrast, the viability of GMneo cells was not altered by NDMA even at concentrations up to 10 mM. Time- and concentration-dependent methylation of DNA, RNA and protein by [14C]NDMA was only observed in cells expressing P450 2E1. Inhibitors of P450 2E1 activity such as ethanol, 4-methylpyrazole, and isoniazid caused a 90% decrease in the methylation of cellular macromolecules and also completely protected the cells against NDMA-mediated toxicity. The cytotoxicity due to exposure to NDMA was partially inhibited by antioxidants such as N-acetylcysteine, ascorbic acid, butylated hydroxyanisole and N-t-butyl-alpha-phenylnitrone but was not potentiated upon glutathione depletion. These results document the ability of rat P450 2E1 to metabolize NDMA to toxic reactive intermediates and demonstrate that this cell line provides a useful model for studying the mechanisms of metabolism-mediated toxicity and carcinogenesis.      

The min mouse on FVB background: susceptibility to spontaneous and carcinogen-induced intestinal tumourigenesis

The multiple intestinal neoplasia (Min) mouse is a model for intestinal tumourigenesis. On the C57BL/6J (B6) background, with an incidence of 100%, the Min mouse develops numerous tumours throughout the intestine, particularly in the small intestine. The Min phenotype was backcrossed to the FVB/NJ (FVB) genetic background in order to reduce the number of tumours. Control FVB Min mice had an incidence of 7% tumours, both in the small intestine and in the colon. One or more flat aberrant crypt foci (ACF) were also observed in the colon of 20% of the control mice. Neonatal mice were given one dose of the food-processing contaminant 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in order to test the chemical induction of tumours and flat ACF. Treatment with PhIP significantly increased both the number and incidence of tumours in the small intestine, and gave a non-significant increase of tumours and flat ACF in the colon.     

Alpha-benzene hexachloride exerts hormesis in preneoplastic lesion formation of rat hepatocarcinogenesis with the possible role for hepatic detoxifying enzymes

Recently there has been a shift in the prevailing paradigm regarding the dose dependence of carcinogen action with increasing acceptance of hormesis phenomenon, although underlying mechanisms remain to be established. To ascertain whether alpha-benzene hexachloride (alpha-BHC) might act by hormesis, rats were initiated with diethylnitrosamine and then alpha-BHC ranging from 0.01 to 500 ppm was administered in the diet for 10 weeks. The highest concentration of alpha-BHC significantly increased the number and area of glutathione S-transferase placental form (GST-P) positive foci, preneoplastic lesions in the liver, but its low dose, 0.05 ppm, caused significant reduction, showing a J-shape dose-response curve. The proliferating cell nuclear antigen positive index for GST-P positive foci in the low dose-treated group was significantly reduced. The dose response curves of CYP450 content, NADPH-P450 reductase activity and 8-hydroxydeoxyguanosine formation revealed the same pattern as GST-P positive foci data. The response curves of CYP2B1 and 3A2 in their activities, protein and mRNA expression showed a threshold but CYP2C11 activity exhibited an inverted J-shape. These results might suggest the possibility of hormesis of alpha-BHC at early stages of rat hepatocarcinogenesis. The possible mechanism involves induction of detoxifying enzymes at low dose, influencing free radical production and oxidative stress, and consequently pathological change in the liver.     

Fractionation sensitivities and dose-control relations of head and neck carcinomas: analysis of the randomized hyperfractionation trials.

PURPOSE: A therapeutic benefit can be achieved by hyperfractionation (HF) if tumours have small fractionation sensitivities characterized by alpha/beta values greater than those for late effects of dose limiting normal tissues. It is the purpose of the present paper to estimate alpha/beta values for head and neck carcinomas from randomized HF trials. MATERIALS AND METHODS: Maximum likelihood estimates the alpha/beta ratio were obtained from tumour control data from the randomized HF trials using the LQ model and a logit or probit type dose-response curve. A joint analysis of five randomized HF trials for head and neck carcinomas was performed to estimate overall alpha/beta and gamma50 values for tumour control. In addition, alpha/beta ratios for the individual trials were estimated using fixed gamma50 values (characteristic quantifying the steepness of dose-response curves) between 1.4 and 5 for tumours. RESULTS: An overall gamma50 of 3.1 (1.5-4.7) was estimated for the dose-tumour control relation from the HF trials, assuming a logit or probit dose-response curve. The tumours showed small fractionation sensitivities characterized by an overall alpha/beta of 10.5 (6.5-29) Gy. One trial allowed quantitative estimation of the alpha/beta values for late normal tissue damage: The alpha/beta estimate for late effects of grade 2 + was 4.0 (3.3-5.0) Gy, assuming a fixed gamma50 of 5 and was even smaller for smaller gamma50 values. CONCLUSION: Head and neck carcinomas showed small fractionation sensitivities with alpha/beta values greater than those typical for bone, soft tissues, and skin, as well as steep dose response curves. Thus, important prerequisites for improving the therapeutic benefit of radiotherapy of head and neck carcinomas by HF are fulfilled for patients who met the accession criteria of the trials.     

Effects of a high fat diet on liver DNA methylation in rats exposed to N-nitrosodimethylamine

Previous experiments have shown that a high fat diet changes incidence and tumour sites by N-nitroso-dialkylamines. The purpose of this study was to examine the effect of high and low fat diet on DNA methylation 6 weeks after the end of a chronic N-nitrosodimethylamine (NDMA) exposure (total dose 150 mg/kg). The concentration of O6-methyldeoxyguanosine (O6-MedG) in liver DNA was measured by immunoassays. The level of O6-MedG persisted 6 weeks after the last dose of NDMA and was 6-fold higher (P less than 0.05) in animals on high fat as compared to low fat diet. In another experiment, in which rats on a low and high fat diet received a single NDMA dose (2 mg/kg), the time-dependent removal of O6-MedG from liver and the hepatic O6-methylguanine DNA-alkyltransferase activity was not modified by the type of diet. These results indicate that a high fat diet enhances DNA methylation in the liver, after chronic treatment by NDMA, and that this effect is likely to be responsible for an increased incidence of liver haemangiosarcomas.     

A simple dose-response relationship for modeling secondary cancer incidence after radiotherapy

Estimates of secondary cancer risk after radiotherapy are becoming more important for comparative treatment planning. There is great uncertainty concerning the dose-response relationship for radiation-induced carcinogenesis at doses higher than 4 Gy. The purpose of this report is to determine a simple dose-response relationship for secondary cancer incidence after radiotherapy treatment which can be used for comparative treatment planning. In this report a simple one-parameter model to estimate the complication probability of secondary cancer was fitted to literature data on secondary cancer incidence after radiotherapy. The results showed a linear dose-response relationship in the low-dose part and an exponentially decreasing one after a maximum at around 10 Gy. The observed dose-response relationship and the literature data used to fit the dose-response indicate that cell death effects are important for the explanation of secondary cancer incidence. Even using a dose and dose-rate effectiveness factor (DDREF) of two (instead of one), a cancer incidence maximum is observed at around 10 Gy, with decreasing incidence at higher doses.     

Analysis of the inhibition of N-nitroso-dimethylamine activation in the liver by N-nitro-dimethylamine using a new non-linear statistical method

N-nitro-dimethylamine (NTDMA) is carcinogenic to rats: it induces nasal cavity tumours. It can be demethylated to N-nitromethylamine and formaldehyde and reduced to N-nitroso-dimethylamine (NDMA): a potent liver carcinogen and also of the nasal cavity if activation in the liver is blocked. To explain the mechanism of NTDMA carcinogenicity we compared its demethylation with that of NDMA in liver microsomes from female and male rats, untreated, fasted or treated with ethanol to induce cytochrome P450 2E1 (CYP2E1). Kinetic parameters were analysed by nonlinear statistical methods, which yielded unbiased parameter estimates for the calculated Km and Vmax values. Km for both compounds was very similar in females (24-47 microM) whereas Vmax for NTDMA was consistently higher than for NDMA as substrate: 1.07-4.70 nmol formaldehyde/mg microsomal protein x min and 0.52-2.76 nmol, respectively. In liver microsomes from induced male rats NTDMA was found to be a much more effective inhibitor of NDMA activation (KEI 39.6-73.6 microM) than NDMA of NTDMA demethylation (KEI 224-286 microM). Nasal microsomes can demethylate both NDMA and NTDMA but the kinetics are vastly different. NTDMA is demethylated at a linear rate and approximately 10-fold more effectively than NDMA. The mechanism of carcinogenicity of ingested NTDMA, we propose, is a partial reduction to NDMA in the liver and inhibition of NDMA activation in the liver by residual NTDMA, which enables NDMA to reach the nasal mucosa where it is activated to DNA-alkylating species and the observed tumours are formed.     

Effects of a single dose of polychlorinated biphenyls to infant mice on N-nitrosodimethylamine-initiated lung and liver tumors

Polychlorinated biphenyls (PCBs) are widespread, chemically-stable environmental contaminants; some congeners are commonly found in human adipose tissue and breast milk. We investigated the effects of a single dose of one PCB mixture (Aroclor 1254) on tumors initiated by N-nitrosodimethylamine (NDMA), also a common environmental agent. Infant outbred Swiss male mice were treated with NDMA (5 mg/kg) i.p. on the 4th day of life, to initiate lung and liver tumors. Four days later each received a single intragastric dose of PCBs (50, 250, or 500 mg/kg of Aroclor 1254) or oil. Groups were killed 16 and 28 weeks later. At both endpoints the mice given 500 mg/kg PCBs after NDMA developed twice as many lung tumors (alveologenic adenomas) as those treated with NDMA only, a significant difference. The PCBs alone did not cause lung tumors. This is the first demonstration of tumor promotion by PCBs in an extrahepatic organ, and it occurred after a single exposure. There were also complex, multiple effects on NDMA-caused liver tumors (adenomas and carcinomas) and on focal hepatocellular proliferative lesions: PCB treatment after the NDMA was associated with decreased number but increased size of these tumors and foci. All of these changes were accompanied by retention in the bodies of 0.1-6 ppm PCBs, as indicated by gas chromatography with electron capture detection. Of this, 80% or more consisted of 2,4,5,2',4',5'-and 2,3,4,2',4',5'-hexachlorobiphenyls in about equal amounts for periods up to 28 weeks. These results point to a need for both experimental and epidemiological studies of the effect of PCB body burden on tumor development.     

Pharmacokinetics of N-nitrosodimethylamine in beagles

The pharmacokinetics of N-nitrosodimethylamine (NDMA) has been studied in beagles. Four male beagles were given 0.5- and 1.0-mg/kg doses of NDMA i.v. and 1.0- and 5.0-mg/kg doses p.o., and at appropriate times after dosing blood samples were drawn and the concentration of NDMA was measured. The experiments were separated by at least 1 week. Following a bolus i.v. dose, the concentration of NDMA in blood declined biphasically with a mean distribution half-life of 19 min and a mean elimination half-life of 73 min. The areas under the blood concentration versus time curves were proportional to the dose indicating that the pharmacokinetics in this dose range were first order. The mean systemic clearance was 43.3 ml/min/kg, the volume of distribution at steady state was 1.9 liters/kg and the mean residence time was 45 min. The clearance of NDMA in the dog was entirely metabolic because no NDMA could be detected in urine after i.v. dosing. The areas under the curve and maximum concentration in blood after the two p.o. doses were not proportional to dose. The evidence suggests that the pharmacokinetics of the 1.0-mg/kg dose were first order, but at the 5.0-mg/kg dose the metabolism of NDMA was saturated. The bioavailability of the lower p.o. dose (i.e., the fraction of the dose that reached the systemic circulation) averaged 93%. The high bioavailability was unexpected since, in the rat, the bioavailability of NDMA is only about 10%, and the systemic clearance in the dog exceeds hepatic blood flow. These data suggest that a substantial fraction of the systemic clearance is extrahepatic and that the pharmacokinetics of NDMA in higher species may be quite different from that observed in rodents.     

The pharmacologic basis of high dose chemotherapy with haematopoietic stem cell support for solid tumours

The theoretical basis of high dose chemotherapy with haematopoietic stem cell support (HDT) for solid tumours is the presumption of a linear, steep dose-response relationship for chemotherapy conditioning agents. We review preclinical pharmacologic studies evaluating steep dose-response relationships for different chemotherapeutic agents, identified through a MED-LINE and CANCER-LIT search of the English medical literature from January 1966 to December 1999. Only BCNU, melphalan, nitrogen mustard, and the combination of thiotepa and cyclophosphamide demonstrated steep dose-response relationships over a wide dose-range. The pharmacologic evidence for the use of other antineoplastic agents for HDT in solid tumours is non-existent. More preclinical studies are needed for a rational development of this therapeutic approach for solid tumours.