Lack of initiation activity in rat liver of low doses of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

It has been generally accepted that genotoxic carcinogens have no threshold in exerting their potential for cancer induction. However, the non-threshold theory can be challenged for cancer risk assessment in humans. Here we examined low dose carcinogenicity of a food-derived, genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), using an in vivo medium-term bioassay to detect initiating activity for rat hepatocarcinogenesis. With MeIQx initiation at various doses followed by administration of phenobarbital, a well known hepatopromoter, no induction of glutathione S-transferase placental form-positive foci, assessed as preneoplastic lesions, was noted at doses of 0.001-1 ppm. The results imply a no-observed effect level for hepatocarcinogenicity with this genotoxic agent.     

The effect of dose and cytochrome P450 induction on the metabolism and disposition of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx) in the rat

The effect of dose and cytochrome P450 induction on the metabolism and disposition of the food-borne carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was investigated in the male Sprague-Dawley rat. Animals were given MeIQx by gavage at doses of 0.01, 0.2 or 20 mg/kg body wt. The phase II conjugates, MeIQx-N2 sulfamate and MeIQx-N2 glucuronide were the predominant metabolites found in urine of non-induced animals at the highest dose treatment. Animals induced with polychlorinated-biphenyl (PCB) produced greater amounts of metabolites hydroxylated at the 5 position of MeIQx which were excreted as glucuronide or sulfate conjugates. At the lowest dose studied, the urinary excretion profile was nearly identical for both animal groups and cytochrome P450 induction had little influence on metabolism. In contrast to high dose exposure, where sulfamate formation was a major route of detoxification, N2 glucuronide formation was the most important metabolic pathway for elimination of MeIQx at low doses. Liver microsomes transformed MeIQx to the genotoxic metabolite 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline (HNOH-MeIQx) and N-hydroxylase activity was 20-fold greater in microsomes obtained from PCB-treated animals than in untreated control animals. The increase in N-hydroxylase activity was discerned in vivo through formation of the metastable N-glucuronide conjugate of HNOH-MeIQx (MeIQx-[HO-N]-Gl). This metabolite accounted for approximately 3% of the dose in bile of PCB-treated rats. In contrast, in the non-induced rat, MeIQx-[HO-N]-Gl was preferentially excreted in urine and accounted for approximately 0.2-1% of the total dose. These results demonstrate that the metabolism of MeIQx in the rat is influenced by both dose and cytochrome P450 induction. The absence of intestinal tumors in the non-induced rat may be partially attributed to the low levels of formation and poor biliary excretion of the N-glucuronide conjugate of the genotoxic metabolite HNOH-MeIQx.     

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.     

Carcinogenicity and mutagenicity of heterocyclic amines in transgenic mouse models.

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein1 promoter-human TGFalpha cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing both the interaction of nuclear oncogenes and growth factors in tumorigenesis. In addition, these mice provide an experimental model to test how environmental chemicals might interact with the c-myc and TGFalpha transgenes during the neoplastic process. Treatment of the double transgenic mice with both genotoxic agents such as diethylnitrosamine and 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) as well as the tumor promoter phenobarbital greatly accelerated the neoplastic process. To investigate the role of mutagenesis in the carcinogenic process, 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) induced mutagenesis and hepatocarcinogenicity was examined in C57BL/lacZ (Muta Mice) and double transgenic c-myc/lacZ mice that carry the lacZ mutation reporter gene. The MelQx hepatocarcinogenicity was associated with an increase in in vivo mutagenicity as scored by mutations in the lacZ reporter gene. These results suggest that transgenic mouse models may provide important tools for testing both the carcinogenic potential of environmental chemicals and the interaction/cooperation of these compounds with specific genes during the neoplastic process.     

Oxidative DNA and RNA damage in rat liver due to acetoxime: similarity to effects of 2-nitropropane.

Acetoxime (ACO) and 2-nitropropane (2-NP), both industrially important chemicals and known hepatocarcinogens in rats, induced increased levels of 8-hydroxy-guanine in liver DNA and RNA of male Sprague-Dawley and F344 rats after either oral or i.p. administration. Both compounds also produced qualitatively the same patterns of other apparent modifications of liver DNA and RNA nucleosides, discernible by HPLC with electrochemical detection. Six hours after administration, the effects of 2-NP on liver nucleic acids were more pronounced in F344 rats than in Sprague-Dawley rats, suggesting that 2-NP may prove to be a stronger carcinogen in the F344 strain. The effects of ACO, a weaker carcinogen than 2-NP, were less than those of the nitroalkane in both rat strains. These results suggest that the hepatocarcinogenicity of ACO, like that of 2-NP, may depend on increased generation of reactive oxygen species capable of producing DNA and RNA base damage in rat liver. In addition, the data support the hypothesis that the hepatocarcinogenicity of ACO depends on its partial in vivo N-oxidation to 2-NP.     

Existence of a no effect level for MeIQx hepatocarcinogenicity on a background of thioacetamide-induced liver damage in rats

As exposure to heterocyclic amines might increase the risk of liver cancer, we investigated the carcinogenic potential of MeIQx under conditions of liver damage caused by TAA. Male, 6-week-old F344 rats (n = 280) were divided into 14 groups; groups 1-7 received TAA (0.03% in drinking water) and groups 8-14 received water for the first 12 weeks. Thereafter, the animals received MeIQx at doses from 0, 0.001, 0.01, 0.1, 1, 10 to 100 p.p.m. (groups 1-7 and 8-14, respectively) in pellet basal diet for 16 weeks. All survivors were killed at week 28 for assessment of numbers and areas of GST-P positive foci, considered to be pre-neoplastic lesions of the liver. Values were increased significantly in all the groups receiving TAA-->MeIQx compared to MeIQx alone (P < 0.01). Numbers of GST-P positive foci were significantly increased in groups 7 and 14 (treated with 100 p.p.m. MeIQx) as compared to 0 p.p.m.-MeIQx (groups 1 and 8) (P < 0.01), along with areas in group 14 compared to group 8 (P < 0.01). However, with the maximum likelihood method, the data for numbers of GST-P positive foci (groups 1-7 and groups 8-14) fitted the hockey stick regression model, representing no differences from groups 1-5 and from groups 8-13, despite a linear dose-dependent increase of MeIQx-DNA adducts from 0.1 to 100 p.p.m. We conclude that there is a no effect level for MeIQx hepatocarcinogenicity, even on a background of TAA-induced liver damage.     

Low-dose carcinogenicity of a heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline: relevance to risk assessment

Male, 21-day-old, F344 rats were administered 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in the diet at various low doses and a high dose, 100 ppm for 16 weeks. Quantitative values for glutathione-S-transferase placental form (GST-P)-positive foci in their livers were similar among the 0, 0.001, 0.01, 0.1 and 1 ppm MeIQx group while 10 ppm MeIQx administration slightly and 100 ppm MeIQx significantly increased their numbers. These results indicate that MeIQx has a no-observed effect level for induction of preneoplastic lesions in rats. Transplacental and trans-breast milk exposure to low doses of MeIQx also did not exert carcinogenic potential in F344 rats and 20% of calorie restriction clearly inhibited development of GST-P-positive foci. The results are of direct significance to human risk assessment.     

The measurement of MeIQx adducts with mouse haemoglobin in vitro and in vivo: implications for human dosimetry

We have investigated covalent binding of radiolabelled [14C]2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) to mouse haemoglobin in vitro and in vivo. Furthermore, we report the development of a capillary column gas chromatography negative ion mass spectrometry (GC-MS) assay capable of detecting MeIQx liberated from haemoglobin after acid or base hydrolysis. Following microsomal activation, the amount of radiolabelled material associated with haemoglobin in vitro increased with incubation time to 0.67 +/- 0.15 nmol/mg haemoglobin at 2 h (initial concentration 0.47 mM [14C]MeIQx, mean +/- SD, n = 6). Hydrolysis of these samples with acid revealed that 47-60% of the radiolabelled material covalently bound to haemoglobin was acid labile. Of this, 7.2-9.8% was recovered as MeIQx as determined by GC-MS. This liberated fraction should reflect the amount of sulphinic acid amide present which is formed when N-hydroxy-MeIQx reacts with sulphydryl-containing amino acids present in haemoglobin. In vivo, no radiolabelled material bound to haemoglobin could be detected in animals treated with the lowest dose of MeIQx (0.2 mg/kg). At higher doses, there was a dose-dependent increase in the covalent binding of radiolabel to haemoglobin (2.0-200 mg/kg). However, the GC-MS assay for hydrolysable adducts of MeIQx yielded detectable quantities of MeIQx (32.2 +/- 17.5 fmol MeIQx/mg haemoglobin) only at the highest dose used. Application of the GC-MS assay to human haemoglobin samples showed that acid-labile adducts of MeIQx, if present, were below the limit of detection of the assay. These results show that levels of sulphinamide adducts of the dietary aromatic amine MeIQx, with haemoglobin, are very low and the implications for future human dosimetry of this carcinogen are discussed.     

Frequency of Ha-ras-1 gene mutations inversely correlated with furan dose in mouse liver tumors

Liver tumors were induced in infant male B6C3F1 mice by preweaning administration of furan, either as a single dose of 400 mg/kg body weight or six doses of 200 mg/kg body weight. Six doses of 200 mg/kg furan resulted in an increased incidence and multiplicity of liver tumors relative to the multiple-dose vehicle control group and the single-dose furan group. In the single-dose group, there was an increase in overall tumor multiplicity but not prevalence of liver tumors. After polymerase chain reaction amplification of isolated DNA, slot-blot oligonucleotide hybridization was used to screen for mutations at known mutational hot-spots in the first three exons of Ha-ras-1 (Hras1). The relative frequency of Hras1 activation was 82% in the 28 tumors analyzed from the single-dose group and 32% in the 28 tumors analyzed from the multiple-dose group. The lack of histomorphologic evidence of chronic cytotoxicity in the livers and the pattern of Hras1 activation suggest that a genotoxic mode of action is responsible for at least part of the hepatocarcinogenicity of furan in B6C3F1 mice. These findings confirm previously documented hepatocarcinogenicity of furan in B6C3F1 mice and provide evidence that carcinogen dose may influence the frequency of Hras1 activation in mouse liver tumors. Mol. Carcinog. 18:199–205, 1997. © 1997 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.

     

Down-regulation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced CYP1A2 expression is associated with bovine lactoferrin inhibition of MeIQx-induced liver and colon carcinogenesis in rats.

The inhibitory influence of bovine lactoferrin (bLF) on induction of preneoplastic hepatic glutathione S-transferase placental form-positive (GST-P( +)) cell foci and colon aberrant crypt foci (ACF) by diethylnitrosamine (DEN) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was investigated in F344 rats. Rats were initially treated with DEN, then placed on basal diet containing MeIQx (200 ppm) alone, MeIQx plus 2% bLF, or MeIQx plus 0.2% bLF from week 2 to week 8, with partial hepatectomy performed at week 3. Concomitant administration of 2% or 0.2% bLF with MeIQx caused significant dose-dependent decreases in both number and unit area of GST-P(+) cell foci (2% bLF, P < 0.001; 0.2% bLF, P < 0.01). Similar results were observed for MeIQx-induced colon ACF in the groups without DEN treatment (2% and 0.2% bLF, P < 0.05). To investigate the underlying mechanisms, we analyzed the influence of bLF on levels of cytochrome P4501A2 (CYP1A2), a metabolically activating enzyme of MeIQx in the liver. The results demonstrated that combined administration of 2% bLF significantly reduced levels of MeIQx-induced CYP1A2 mRNA (P < 0.05) and protein (P < 0.05) to the normal levels, in association with reduced values for MeIQx-DNA adducts (P < 0.05), liver GST-P(+) cell foci and colon ACF. These results suggest that bLF is a chemopreventive agent for DEN alone or DEN plus MeIQx-induced liver, and MeIQx-induced colon carcinogenesis in rats. One possible mechanism is a normalizing down-regulation of CYP1A2 expression by bLF, with consequent reduction of carcinogen activation and adduct formation.     

Chronic ethanol ingestion and the hepatocarcinogenicity of N-hydroxy-N-2-fluorenylacetamide

The effect of long-term administration of 10% and 20% ethanol solutions on the hepatocarcinogenicity of N-hydroxy-N-2-fluorenylacetamide in two strains of rats was investigated. In tests with ethanol and carcinogen, started with 4- to 6-week-old Fischer rats the size of the liver and the extent of tumor formation were slightly less with male rats, and slightly more with females, as compared to rats fed only carcinogen. Experiments beginning with 16-week-old males, or with ethanol following carcinogen, were without additional effect. In NIH black rats, less susceptible to carcinogen and with a longer experimental period of 64 weeks, the concurrent intake of 10% ethanol did not affect the liver tumor incidence, although in females there seemed to be fewer cholangiomas and more hepatomas.     

Effects of Chronic Administration of Low Doses of 2-Ammo-3,8-dimethylimidazo-[4,5-f]qumoxaline on Glutathione S-Transferase Placental Form-positive Foci Development in the Livers of Rats Fed a Choline-deficient Diet

Effects of chronic administration of 2-amino-3,8-dimethylimidazo[4,S- f ]quinoxaline (MeIQx) at the very low doses of 0.4 and 4 ppm, respectively 1000- and 100-fold less than the dose shown to be carcinogenic (400 ppm), on the liver of rats fed a choline-deficient (CD) diet were examined in terms of glutathione S-transferase placental form (GST-P)-positive foci. Male F344 rats were given CD diet containing 0, 0.4 or 4 ppm MeIQx for 20 or 40 weeks. As controls, rats received choline-supplemented (CS) diet in the same manner. MeIQx at 4 ppm in the CD diet significantly increased both the number and area of GST-P-positive foci, the values being 2.3- and 2.1-fold at 20 weeks and 2.0- and 3.3-fold at 40 weeks, respectively, compared with those observed for CD diet alone. MeIQx at 0.4 ppm in CD diet did not affect the development of GST-P-positive foci. No influence of the heterocyclic amine was found in the CS groups, where only very small numbers of minute lesions were observed. The level of MeIQx-DNA adducts in rats given the CD diet containing 4 ppm MeIQx was 2- to 3-fold lower than that in rats given the CS diet containing 4 ppm MeIQx at 20 and 40 weeks. This result indicates that DNA adduct formation and cell proliferation are both required for the increase of GST-P-positive foci in rats fed 4 ppm MeIQx in a CD diet. The above findings strongly suggest that MeIQx could be carcinogenic even at 4 ppm under CD conditions, where liver cell regeneration is continuously occurring.     

Hepatocellular Carcinoma Induction in LEC Rats by a Low Dose of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline

A food-borne heterocyclic amine, 2-amino-3,8-dimethylimidazo[4,5- f ]qninoxaline (MeIQx), induces hepatocellular carcinomas (HCCs) in F344 male rats at an incidence of 95%, when fed in the diet at 400 ppm for 61 weeks. In this study, the effect of a low dose of MeIQx was examined in Long-Evans with cinnamon-like coat color (LEC) rats, which have a mutation in Atp7b and suffer from hereditary hepatitis and HCCs, with high levels of copper accumulation in the liver. Rats of the LEC and Long-Evans with agouti coat color (LEA) sibling lines were given a diet containing 40 ppm MeIQx from the age of 23 weeks to 63 weeks, for a total administration period of 40 weeks. In LEC rats, HCCs were observed in 8/8 animals administered MeIQx, and 2/8 rats receiving a normal diet. The number of HCCs per rat (mean±SD) was 2.8±2.0 and 0.3±0.5, respectively. In the LEA rats, however, no tumors were induced by administration of MeIQx. These results indicate that damaged liver associated with compensatory cell proliferation is much more susceptible to chemical hepatocarcinogens, including MeIQx, than the normal liver.     

Down-regulation of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced CYP1A2 Expression Is Associated with Bovine Lactoferrin Inhibition of MeIQx-induced Liver and Colon Carcinogenesis in Rats

The inhibitory influence of bovine lactoferrin (bLF) on induction of preneoplastic hepatic glutathione S -transferase placental form-positive (GST-P⁺) cell foci and colon aberrant crypt foci (ACF) by diethylnitrosamine (DEN) and 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx) was investigated in F344 rats. Rats were initially treated with DEN, then placed on basal diet containing MeIQx (200 ppm) alone, MeIQx plus 2% bLF, or MeIQx plus 0.2% bLF from week 2 to week 8, with partial hepatectomy performed at week 3. Concomitant administration of 2% or 0.2% bLF with MeIQx caused significant dose-dependent decreases in both number and unit area of GST-P⁺ cell foci (2% bLF, P <0.001; 0.2% bLF, P <0.01). Similar results were observed for MeIQx-induced colon ACF in the groups without DEN treatment (2% and 0.2% bLF, P <0.05). To investigate the underlying mechanisms, we analyzed the influence of bLF on levels of cytochrome P4501A2 (CYP1A2), a metabolically activating enzyme of MeIQx in the liver. The results demonstrated that combined administration of 2% bLF significantly reduced levels of MeIQx-induced CYP1A2 mRNA ( P <0.05) and protein ( P <0.05) to the normal levels, in association with reduced values for MeIQx-DNA adducts ( P <0.05), liver GST-P⁺ cell foci and colon ACF. These results suggest that bLF is a chemopreventive agent for DEN alone or DEN plus MeIQx-induced liver, and MeIQx-induced colon carcinogenesis in rats. One possible mechanism is a normalizing down-regulation of CYP1A2 expression by bLF, with consequent reduction of carcinogen activation and adduct formation.     

Low doses and thresholds in genotoxicity: from theories to experiments.

The absence of threshold in the action of genotoxic carcinogens was theoretically postulated more than thirty years ago, but continuously challenged for scientific and practical reasons. The direct experimental demonstration of the presence of a threshold for genotoxic damage is precluded by the insufficient sensitivity of the biological methods presently available. In the last twenty years the sensitivity of the methods for quantitative determination of the DNA adducts of the carcinogens was enormously improved, demonstrating linearity of the dose/adducts pattern over dose intervals of more than million-fold. The arguments more often advanced for the presence of a threshold for genotoxic carcinogens were mainly based on the action of intracellular scavengers, detoxification enzymes and repair systems, being able to block completely the genotoxic carcinogens at very low doses. This hypothesis is disproved by the constant presence of DNA adducts at extremely low doses of different carcinogens, whatever their chemical structure can be. On the other hand if genotoxic damage results from damage to proteins involved in cell division, like tubulin, there is a threshold dose for such genotoxic effects. The detailed knowledge of the genotoxicity mechanism is therefore needed for a sound carcinogenic risk assessment. Most of the genotoxic carcinogens, or their metabolites, damage directly the DNA. In this case the absence of threshold must be assumed, not only for theoretical reasons, but for the results of the experiments quantitatively relating DNA damage and very low doses of carcinogens. For the sake of clarity the "adjectivated" thresholds, like practical pragmatic, apparent and operational, must disappear from documents analysing the carcinogenic risk.     

In vivo mutagenicity and hepatocarcinogenicity of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in bitransgenic c-myc/lambda lacZ mice.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a mutagenic and carcinogenic heterocyclic amine found in cooked meat. Hepatic DNA adduct formation, in vivo mutagenicity, and hepatocarcinogenicity of MeIQx were examined in mice harboring the lacZ mutation reporter gene (Muta mice) and bitransgenic mice overexpressing the c-myc oncogene. C57Bl/lambda lacZ and bitransgenic c-myc (albumin promoter)/lambda lacZ mice were bred and weaned onto an American Institute of Nutrition-76-based diet containing 0.06% (w/w) MeIQx or onto control diet. After 30 weeks on diet, only male bitransgenic mice on MeIQx developed hepatocellular carcinoma (100% incidence). By 40 weeks, hepatic tumor incidence was 100%/75% (17%/0%) and 44%/17% (0%/0%) in male c-myc/lambda lacZ and C57Bl/lambda lacZ mice who were given MeIQx (or control) diet, respectively, supporting a synergism between MeIQx and c-myc overexpression in hepatocarcinogenesis. At either time point, mutant frequency in the lacZ gene was at least 40-fold higher in MeIQx-treated mice than in control mice of either strain. These findings suggest that MeIQx-induced hepatocarcinogenesis is associated with MeIQx-induced mutations. Elevated mutant frequency in MeIQx-treated mice also occurred concomitant with the formation of MeIQx-guanine adducts, as detected by the 32P-postlabeling assay. Irrespective of strain or diet, sequence analysis of the lacZ mutants from male mouse liver showed that the principal sequence alterations were base substitutions at guanine bases. Adenine mutations, however, were detected only in animals on control diet. MeIQx-fed mice harboring the c-myc oncogene showed a 1.4-2.6-fold higher mutant frequency in the lacZ gene than mice not carrying the transgene. Although there was a trend toward higher adduct levels in c-myc mice, MeIQx-DNA adduct levels were not significantly different between c-myc/lambda lacZ and C57Bl/lambda lacZ mice after 30 weeks on diet. Thus, it seemed that factors in addition to MeIQx-DNA adduct levels, such as the enhanced rate of proliferation associated with c-myc overexpression, may have accounted for a higher mutant frequency in c-myc mice. In the control diet groups, the lacZ mutant frequency was significantly higher in c-myc/lambda lacZ mice than in C57Bl/lambda lacZ mice. The findings are consistent with the notion that c-myc overexpression is associated with an increase in mutagenesis. The mechanism for the synergistic effects of c-myc overexpression on MeIQx hepatocarcinogenicity seems to involve an enhanced expression of MeIQx-induced mutations.     

Dose-response relationship and low dose extrapolation in chemical carcinogenesis

Data supporting various dose-response relationships in chemical carcinogenesis are summarized. General principles are derived to explain the relationships between exposure dose, DNA adduct level, induction of genetic changes, and tumor incidence. Some mechanistic aspects of epigenetic carcinogens (stimulation of cell division and maldifferentiation) are analyzed in a similar way. In a homogeneous population, non-linearities are frequent. They are due to phenomena of induction or saturation of enzymatic activities and to the multi-step nature of carcinogenesis: if a carcinogen accelerates more than one step, the superposition of the dose-response curves for the individual steps can result in an exponential relationship. A fourth power of the dose was the maximum seen in animals (formaldehyde). At the lowest dose levels, a proportionality between dose and tumor induction is postulated independent of the mechanism of action if the carcinogen accelerates the endogenous process responsible for spontaneous tumor formation. Low-dose thresholds are expected only for situations where the carcinogen acts in a way that has no endogenous counterpart. Epidemiological studies in humans show linear dose-response curves in all but two investigations. The difference from the strongly nonlinear slopes seen in animal studies could be due to the heterogeneity of the human population: if the individual sensitivity to a carcinogen is governed by a large number of genetic and life-style factors, the non-linearities will tend to cancel each other out and the dose-response curve becomes 'quasi-linear'.     

Comparison of Reversibility of Rat Forestomach Lesions Induced by Genotoxic and Non-genotoxic Carcinogens

Reversibility of forestomach lesions induced by genotoxic and non-genotoxic carcinogens was compared histopathologically. Groups of 30 to 33 male F344 rats were given dietary 0.1% 8-nitroquinoline, dietary 0.4–0.2% 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, an intragastric dose of 20 mg/kg body weight N-methyl-N'-nitro-N-nitrosoguanidine once a week, or 20 ppm N-methylnitrosourethane in the drinking water as a genotoxic carcinogen, or 2% butylated hydroxyanisole, 2% caffeic acid, 2% sesamol or 2% 4-methoxyphenol in the diet as a non-genotoxic carcinogen for 24 weeks. Ten or 11 rats in each group were killed at week 24. Half of the remainder were maintained on basal diet alone for an additional 24 weeks and the other half were given the same chemical for 48 weeks, and then killed. Forestomach lesions induced by genotoxic carcinogens did not regress after removal of carcinogens. In contrast, simple or papillary hyperplasia (SPH), but not basal cell hyperplasia (BCH), induced by non-genotoxic carcinogens clearly regressed after cessation of insult. SFH labeling indices in the non-genotoxic carcinogen-treated cases decreased after removal of the carcinogenic stimulus whereas BCH values were low irrespective of treatment. Atypical hyperplasia (AH), observed at high incidences in rats treated with genotoxic carcinogens, was also evident in animals receiving non-genotoxic agents, even after their withdrawal, albeit at low incidences. AH labeling indices remained high even without continued insult. These results indicate that even with non-genotoxic carcinogens, heritable alterations at the DNA level could occur during strong cell proliferation and result in AH development. This putative preneoplastic lesion might then progress to produce carcinomas.     

New insights into carcinogenesis of the classical model arylamine 2-acetylaminofluorene.

2-Acetylaminofluorene (AAF) is a complete carcinogen in rat liver. The genotoxic effects of reactive metabolites are considered necessary but not sufficient to explain tumor formation. An overview is given of an AAF-feeding experiment designed to demonstrate early effects, preceding the development of enzyme-altered foci to support the hypothesis that toxic effects lead to a cirrhosis-like transformation as a prerequisite for the expansion of initiated foci and how those effects influence the dose-time-response relationship of tumor formation. Male Wistar rats were fed 0.005, 0.01, 0.02, 0.04 and 0.08% AAF in the diet for 2, 4, 8, and 16 weeks. GST-P-positive foci developed more than proportionately only at 16 weeks. As a first sign of morphological alterations the number of apoptoses increased (2 weeks), the proliferation rate followed with some delay and was maximal at 4 weeks. The most sensitive parameter for adaptive responses was the inhibition of the mitochondrial permeability transition, studied ex vivo. All parameters increased dose-dependently at low doses. A threshold could not be detected, but effects developed much more gradually with the lowest, non-toxic dose. The situation of massive development of foci observed with the higher doses at 16 weeks was not reached. Apoptosis and proliferation rate reach a plateau between 4 and 8 weeks with some of the doses indicating a period in which some balance between adaptation and stress response exists.     

Enhancement by Cigarette Smoke Exposure of 2-Amino-3,8-dimethylimidazo[4,5- f]quinoxaline-induced Rat Hepatocarcinogenesis in Close Association with Elevation of Hepatic CYP1A2

The modifying effects of cigarette smoke (CS) exposure on a heterocyclic amine (HCA) 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx)-induced carcinogenesis were investigated in male F344 rats. Groups 1 and 2 were fed MeIQx at a dose of 300 ppm, and simultaneously received CS and sham smoke (SS) for 16 weeks, respectively. Groups 3–5 were given the MeIQx diet for 4 weeks, and simultaneously exposed to CS for 4 weeks (group 3), exposed to CS for 12 weeks after the MeIQx treatment (group 4) or received SS for 16 weeks (group 5). Groups 6 and 7 were fed basal diet and respectively received CS and SS for 16 weeks. In terms of the mean number or area, the development of glutathione S-transferase placental form-positive (GST-P⁺) liver cell foci was significantly ( P <0.01) greater in group 1 than in group 2. The mean number of colonic aberrant crypt foci (ACFs) per animal was increased by continuous CS exposure regardless of MeIQx feeding, the differences between groups 4 and 5 ( P <0.05), and between groups 6 and 7 ( P <0.05) being significant. Immunoblot analysis confirmed that the hepatic CYP1A2 level in group 6 was remarkably increased as compared to that in group 7. In addition, liver S9 from rats in group 6 consistently increased the mutagenic activities of six HCAs including MeIQx as compared to those in group 7. Thus, our results clearly indicate that CS enhances hepatocarcinogenesis when given in the initiation phase via increasing intensity of metabolic activation for MeIQx and possibly colon carcinogenesis when given in the post-initiation phase in rats induced by MeIQx.