Showing papers in "Carcinogenesis in 1988"

Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke

Abstract: Tobacco-specific nitrosamines are a group of carcinogens that are present in tobacco and tobacco smoke. They are formed from nicotine and related tobacco alkaloids. Two of the nicotine-derived nitrosamines, NNK and NNN, are strong carcinogens in laboratory animals. They can induce tumors both locally and systemically. The induction of oral cavity tumors by a mixture of NNK and NNN, and the organospecificity of NNK for the lung are particularly noteworthy. The amounts of NNK and NNN in tobacco and tobacco smoke are high enough that their total estimated doses to long-term snuff-dippers or smokers are similar in magnitude to the total doses required to produce cancer in laboratory animals. These exposures thus represent an unacceptable risk to tobacco consumers, and possibly to non-smokers exposed for years to environmental tobacco smoke. The permission of such high levels of carcinogens in consumer products used by millions of people represents a major legislative failure. Indeed, the levels of tobacco-specific nitrosamines in tobacco are thousands of times higher than the amounts of other nitrosamines in consumer products that are regulated by government authorities. Although the role of tobacco-specific nitrosamines as causative factors in tobacco-related human cancers cannot be assessed with certainty because of the complexity of tobacco and tobacco smoke, several lines of evidence strongly indicate that they have a major role, especially in the causation of oral cancer in snuff-dippers. Epidemiologic studies have demonstrated that snuff-dipping causes oral cancer. NNK and NNN are quantitatively the most prevalent known carcinogens in snuff, and they induce oral tumors when applied to the rat oral cavity. A role for NNK in the induction of lung cancer by tobacco smoke is likely because of its organospecificity for the lung. Tobacco-specific nitrosamines may also be involved in the etiology of tobacco-related cancers of the esophagus, nasal cavity, and pancreas. Because they are derived from nicotine, and therefore should be associated only with tobacco, tobacco smoke and other nicotine-containing products, tobacco-specific nitrosamines as well as their metabolites and macromolecular adducts should be ideal markers for assessing human exposure to, and metabolic activation of, tobacco smoke carcinogens. Ongoing research has demonstrated the formation of globin and DNA adducts of NNK and NNN in experimental animals. Sensitive methods for the detection and quantitation of these adducts in humans would provide an approach to assessing individual risk for tobacco-related cancers.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of organosulfur compounds from garlic and onions on benzo[a]pyrene-induced neoplasia and glutathione S-transferase activity in the mouse

Abstract: In the present study, eight organosulfur compounds from garlic and onions were studied for their inhibitory effects on benzo[a]pyrene (BP)-induced neoplasia of forestomach and lung of female A/J mice when administered 96 and 48 h prior to carcinogen challenge. These compounds had one, two or three linearly connected sulfur atoms. They included the four allyl group-containing derivatives: allyl methyl trisulfide (AMT), allyl methyl disulfide (AMD), diallyl trisulfide (DAT), and diallyl sulfide (DAS), and also four corresponding saturated compounds in which propyl groups were substituted for the allyl groups. All four allylic compounds inhibited BP-induced neoplasia of the forestomach. The saturated analogs were almost without inhibitory activity, indicating the importance of the allyl groups. DAT, which contains two allyl groups, was more potent than AMT, which contains only one allyl group, thus providing further evidence for the role of allyl groups in the inhibitory effects observed. DAS and AMD, but not DAT or AMT, inhibited pulmonary adenoma formation. The fact that in the lung the monosulfide and disulfide inhibited, but the trisulfide did not inhibit, indicates that the number of sulfur atoms in the molecule can control the organ sites at which protection against carcinogenesis will occur. All four allylic compounds induced increased glutathione S-transferase (GST) activity in the forestomach, but varied in their capacity to induce GST in lung, liver and small bowel. Their saturated analogs produced little or no induction. In evaluating relationships between diet and cancer, it would be useful to consider the possible role of garlic and onion organosulfur compounds as protective agents. In addition, further studies of this class of chemicals might lead to the identification and development of useful new chemopreventive compounds.

Enhancing effect of various hepatocarcinogens on induction of preneoplastic glutathione S-transferase placental form positive foci in rats--an approach for a new medium-term bioassay system.

Abstract: A large series of assays of the hepatocarcinogenic potential of 112 different compounds were carried out using a rapid bioassay system developed in this laboratory based on the two-step concept of hepatocarcinogenesis. Rats were initially given a single dose (200 mg/kg) of diethylnitrosamine (DEN) i.p. and starting 2 weeks later were treated with test compounds for 6 weeks and then killed, all rats being subjected to two-thirds partial hepatectomy (PH) at week 3. Carcinogenic potential was scored by comparing the number and area per cm2 of induced glutathione S-transferase placental form-positive (GST-P+) foci in the liver with those of the corresponding control group given DEN alone. Positive was scored for a significant increase in the value of GST-P+ foci, negative for no change or a decrease. Results were compared to reported Salmonella/microsome and long-term carcinogenicity test findings. Of the liver carcinogens, 10 out of 11 (90.9%) mutagenic, and 11 out of 13 (84.6%) non-mutagenic compounds gave positive results (mean, 87.5%). Carcinogens other than the hepatocarcinogens gave less positive results (two out of 17, 11.8%). None of the compounds reported as non-carcinogenic demonstrated positivity suggesting that the assay system does not suffer from the disadvantage of false-positive results. The protocol system also provided information concerning the inhibitory potential of compounds such as anti-oxidants. It is concluded that the present experimental protocol which requires far fewer animals and shorter duration than a long-term carcinogenicity test has practical applications for the rapid and economical screening of environmental hepatocarcinogens and their inhibitory agents.

Activated neutrophils induce prolonged DNA damage in neighboring cells

Abstract: We have measured the capacity of highly-purified, paraffin oil-elicited neutrophils to induce DNA single-strand breaks in a newly established plasmacytoma cell line, RIMPC 2304, which was induced by a retrovirus containing the c-myc and V-Ha-ras oncogenes. This cell line effectively repairs DNA damage induced by gamma-irradiation. DNA damage induced by neutrophils was correlated with the oxidative burst of the neutrophils. The levels of superoxide anion, H2O2, and HOCl produced after stimulation of the neutrophils (6 X 10(5)/cm3) with the tumor promoter phorbol myristate acetate (100 nM) were 33.8 microM, 12.8 microM and 1.7 microM respectively in 15 min, and 98 microM, 20 microM and 8.7 microM respectively in 90 min. The results of alkaline elution experiments revealed that when the same concentration of neutrophils was co-incubated for 15 min in serum-free medium with an equal number of radioactively labeled RIMPC 2304 cells, the latter incurred a level of damage that approximated that caused by 300 rad equivalents of gamma-irradiation or by a 1-min treatment with 20 microM H2O2 at 37 degrees C. Damage from neutrophils was coincident with the oxidative burst; it was induced rapidly (within 5 min) but remained high for more than 90 min. The level of damage achieved was dependent upon the ratio of neutrophils: target cells and was clearly detectable at ratios as low as 0.25:1. Induction of single-strand breaks was completely inhibited by catalase and partially inhibited by superoxide dismutase, mannitol, and reduced glutathione but not by Na azide. Addition of the non-steroidal anti-inflammatory drug indomethacin either enhanced (at 50 microM) or had no effect (at 2 microM) on the damage detected. Finally, repair of strand breaks induced by neutrophils was significantly slower (half-time approximately 10 min) than that observed for repair of similar levels of damage induced by H2O2 or gamma-irradiation (half-times approximately 3 min, each). The results indicate that neutrophils cause prolonged DNA damage in neighboring cells. Moreover, they indicate that although H2O2 produced in the oxidative burst is an essential mediator of the damage observed, additional reactive oxygen intermediates including the superoxide anion are also implicated. The data are discussed in relation to the possible role of neutrophils in chronic inflammation and in pristane-induced plasmacytoma formation in mice.

Glutathione and glutathione-dependent enzymes in ovarian adenocarcinoma cell lines derived from a patient before and after the onset of drug resistance: intrinsic differences and cell cycle effects

Abstract: The regulation of glutathione and various glutathione-dependent enzymes has been studied in two ovarian adenocarcinoma cell lines derived from a patient before (PE01) and after (PE04) the onset of drug resistance to cis-platinum, chlorambucil and 5-fluorouracil. Reduced glutathione levels were higher in the drug resistant cells (PE04). This could possibly be attributed to a much higher (6.5-fold) gamma-glutamyl-transpeptidase activity. In addition, glutathione-S-transferase (GST) and glutathione peroxidase were 2.9- and 2.3-fold higher in this cell line. Analysis of the GST subunit composition showed both cell lines contained high levels of the acidic GST and lower concentrations of a basic isozyme. The difference in GST activity between PE01 and PE04 did not appear to be related to the levels of these GST subunits. GSH, glutathione peroxidase and gamma-glutamylcysteinyl synthetase were all found to be regulated during the cell cycle, higher levels being detected in logarithmic versus confluent cultures of PE01 and PE04 and MCF7. This did affect some of the differences between PE01 and PE04 and therefore may be a contributing factor to the differential sensitivity of these cells to cytotoxic compounds. The above data provide the first evidence that tumour cells obtained from a patient before and after the onset of drug resistance have significant differences in glutathione-dependent enzyme content.

Inhibition of dietary fat-promoted colon carcinogenesis in rats by supplemental calcium or vitamin D3

Abstract: A 2 X 2 X 2 factorial experimental design was used to investigate the effects of supplemental calcium (Ca) (0.5% versus 1.0% of diet as Ca gluconate) and vitamin D3 (D) (1000 IU/kg diet versus 2000 IU/kg diet) on 1,2-dimethylhydrazine-induced colon carcinogenesis in male F344 rats promoted with a 20% corn oil diet. Animals on the high-fat (HF) diet had an increased tumor incidence compared to the low-fat (LF) control diet (86% versus 53%, P less than 0.05) and supplemental Ca or D reduced this to or below the LF incidence (HF + Ca: 53%, HF + D: 47%). However, supplemental Ca or D had no inhibitory effect with LF diets (LF + Ca: 67%, LF + D: 60%). The results of this study indicate a possible role for supplemental Ca or D in the prevention of colon cancer, effective only in HF diets.

Serum albumin adducts in the molecular epidemiology of aflatoxin carcinogenesis: correlation with aflatoxin B1 intake and urinary excretion of aflatoxin M1

Abstract: Aflatoxin-serum albumin adducts in the blood of 42 residents of Guangxi Province, People's Republic of China, were determined and compared with intake of aflatoxin B1 (AFB1) and excretion of aflatoxin M1 (AFM1) in urine. Blood specimens were obtained during the same period that urine was collected and that diet was sampled. Serum albumin was isolated from blood by affinity chromatography on Reactive Blue 2-Sepharose and subjected to enzymatic proteolysis using Pronase. Immunoreactive products were purified by immunoaffinity chromatography and quantified by competitive radioimmunoassay. A highly significant correlation (r = 0.60, P less than 0.00003) of adduct level with AFM1 excretion was observed. An equally highly significant correlation of adduct level with intake (r = 0.69, P less than 0.000001) was also observed. From the slope of the regression line for adduct level as a function of intake, it was calculated that 1.4-2.3% of ingested AFB1 becomes covalently bound to serum albumin, a value very similar to that observed when rats are administered AFB1.

32P-adduct assay: comparative recoveries of structurally diverse DNA adducts in the various enhancement procedures.

Abstract: A 32P-adduct assay for the measurement of low levels (1 adduct per 10(7) nucleotides) of binding of carcinogens to DNA has been reported previously. In this procedure, DNA is enzymatically hydrolyzed to 3'-monophosphates of normal nucleosides and adducts, which are 5'-32P-labeled by T4 polynucleotide kinase and [gamma-32P]ATP. Labeled adducts are resolved by TLC. Enrichment of adducts by extraction in 1-butanol [Gupta, R.C. (1985) Cancer Res., 45, 5656] or digestion with nuclease P1 [Reddy, M.V. and Randerath, K. (1986) Carcinogenesis, 7, 1543] prior to 32P-labeling, however, increased the sensitivity of detection for many adducts to a level of 1 per 10(9-10) nucleotides, although adduct recovery particularly in the latter assay depended on the chemical nature of adducts. We have now compared recoveries for greater than 70, different carcinogen-DNA adducts of known and unknown chemical nature in the two enrichment procedures as well as in a new procedure in which polynucleotide kinase is substituted for nuclease P1. When compared with the butanol extraction procedure, arylamines (such as 2-aminofluorene, 2-aminophenanthrene, 2-naphthylamine, 4-aminobiphenyl, 4-azoaminobenzene and N'-acetylbenzidine) bound to the C8 position of guanine were lost almost completely (0.2-4% recovery) in the nuclease P1-mediated assay, but the presence of a polar group in the aromatic amine moiety (such as 2-acetylaminofluorene, 2-acetylamino-phenanthrene and methyl-4-azoaminophenyl) rendered similar recovery. In contrast, aromatic amines (2-amino-phenanthrene, 2-acetylaminophenanthrene, 2-acetylaminofluorene and methyl-4-azoaminobenzene) and polycyclic aromatic hydrocarbons (benzo[a]pyrene, bromomethylbenzanthracene and benzanthracene) bound to the exocyclic positions of guanine or adenine showed extensive or as complete recovery in the nuclease P1 procedure as in the extraction procedure. Some of the unknown presumably polar adducts showed a lower recovery (30-70%) in the butanol procedure as compared to the nuclease P1 enrichment. The recovery pattern of most adducts examined in the polynucleotide kinase-enrichment assay was essentially the same as found in nuclease P1-mediated assay, except that overall lower values were obtained. Our data suggest that a given DNA sample should be analyzed by different versions of the 32P-adduct assay, particularly, DNA of specimens of humans exposed to low levels of unknown carcinogens. The observation that chemical structure of an adduct may be detrimental in its recovery in the enzyme- and extraction-mediated enrichment procedures may serve as a probe in the structural characterization of adducts of unknown carcinogens.

Anti-carcinogenic activity of d-limonene during the initiation and promotion/progression stages of DMBA-induced rat mammary carcinogenesis.

Abstract: d-Limonene was found to be effective in reducing the average number of rat mammary carcinomas that developed in 7,12-dimethylbenz[a]anthracene-treated rats when the terpene was fed during the initiation or during the promotion/progression stage of carcinogenesis. The time to the appearance of the first tumor was extended only when d-limonene was fed during the initiation stage. These effects could not be attributed to changes in mammary-relevant endocrine functions.

In vitro and in vivo inhibitory effect of ethanol and acetaldehyde on O6-methylguanine transferase

Abstract: Human and rat O6-methylguanine transferase (O6MeGT) are inhibited in vitro by ethanol at concentrations of 10 to 50 mM and by acetaldehyde, the first metabolite of ethanol, at concentrations as low as 0.01 microM. Several other enzymes, including glyceraldehyde-3-phosphate dehydrogenase and yeast alcohol dehydrogenase, which like O6MeGT have cysteines in their active sites, were not inhibited by acetaldehyde at the levels that inhibited O6MeGT. Disulfiram, an acetaldehyde dehydrogenase inhibitor, enhanced the inhibitory effect of ethanol in vivo. These results indicate that the inhibitory effect of ethanol on O6MeGT activity is mediated primarily via its metabolite, acetaldehyde.

Continuous cell lines with altered growth and differentiation properties originate after transfection of human keratinocytes with human papillomavirus type 16 DNA.

Abstract: Immortalization of human keratinocytes (HKc) by human papillomavirus type 16 (HPV16) is reproducible at a high frequency, is due directly to the presence of the viral sequences in the cells, and occurs independently from the genetic characteristics of the host cells. Ten human keratinocyte strains, each derived from a different individual, were transfected with pMHPV16d and selected with G418. Eight became established lines. Two strains, which failed to grow shortly after successful G418 selection, were negative for HPV16 DNA. No lines were established following transfection of the same HKc strains with vector sequences only. The immortalized lines maintained a constant number of copies of the viral genome integrated into the cellular DNA. Each line showed a unique integration pattern of HPV16 sequences into the cellular genome, but expressed similar patterns of viral messages. Sublines able to grow in the absence of growth factors (epidermal growth factor and bovine pituitary extract), and others which became resistant to differentiation stimuli (serum and calcium) were obtained by selection in growth factor-free medium and serum-supplemented medium, respectively. The establishment of continuous cell lines is a direct consequence of the presence of viral sequences; however, because none of these lines formed tumors in nude mice, additional events must be necessary for progression of malignancy. HPV16-immortalized human keratinocyte lines can be used to investigate and identify the viral factors involved with the modification of growth and differentiation control by HPV16.

Carcinogenicity in rats of a mutagenic compound, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline.

Abstract: Carcinogenicity of 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx), which is a potent mutagen first isolated from fried beef and widely present in various cooked foods, was tested in both sexes of F344 rats. Rats were continuously given a diet containing 0.04% MeIQx or basal diet and the experiment was finished on day 429. In experimental animals, the incidence of liver, Zymbal gland, clitoral gland and skin tumors was significantly higher than in control animals. The incidence of liver tumors was 100% in males and 53% in females; most liver tumors of males were hepatocellular carcinomas and all liver tumors of females were neoplastic nodules. The incidence of Zymbal gland tumors was 75% in males and 53% in females. Clitoral gland tumors were induced in 63% and skin tumors were observed in 35% of males and 5% of females. Most of these three types of tumors were diagnosed as squamous cell carcinoma. In the control rats, liver, Zymbal gland, clitoral gland and skin tumors were not observed in either sex.

Early signals for keratinocyte differentiation: role of Ca2+-mediated inositol lipid metabolism in normal and neoplastic epidermal cells.

Abstract: Differentiation of cultured keratinocytes is regulated by the Ca2+ concentration of the culture medium. Below 0.1 mM Ca2+, a monolayer of basal cells is formed which fully differentiates in response to a rise in medium Ca2+. A role for protein kinase C in this differentiation program has been suggested because phorbol esters induce epidermal differentiation in cells grown in reduced Ca2+ medium, and exogenously added phospholipase C (which increases cellular diacylglycerol) mimics phorbol ester action. These findings suggested that the external Ca2+ signal may lead to protein kinase C activation via stimulation of cellular phospholipase C activity. The effect of the external Ca2+ signal on phospholipase C was studied in cultures prelabeled with [3H]-inositol. Within 2 min after addition of Ca2+ to 1 mM, an increase in inositol phosphates was measured. This correlated with a decrease in radiolabeled phosphoinositides, suggesting that these were the source of the increased inositol phosphates. After 3 h in 1 mM Ca2+ medium, each of the inositol phosphates remained increased to 130-140% of control levels. Inositol phosphate metabolism in neoplastic epidermal cells was quantitatively similar to normal cells in response to the Ca2+ signal. Stimulation of phosphatidylinositol (PIP) metabolism appears to be mediated by a rise in intracellular free Ca2+ because Ca2+ ionophores A23187 and ionomycin also cause a similar rise in inositol phosphate levels. Phorbol esters did not increase PIP turnover but instead stimulated phosphatidylcholine metabolism. The induction of epidermal differentiation by phorbol esters was enhanced by ionomycin, suggesting that both protein kinase C activation, elevation of intracellular calcium and PIP turnover were important components of the signal for epidermal differentiation. These results demonstrate that the second messenger system for Ca2+-mediated keratinocyte differentiation may be through a direct effect on phospholipase C activity.

β-Carotene and canthaxanthin inhibit chemically- and physically- induced neoplastic transformation in 10T1/2 cells

Abstract: We have studied the effects of beta-carotene (beta-C), a vitamin A precursor of plant origin, and canthaxanthin (CTX), a non-provitamin A carotenoid, on the neoplastic transformation of C3H/10T1/2 murine fibroblast cells. Chemical transformation in this well-characterized cell system has previously been shown to be reversibly inhibited by retinoids, compounds with vitamin A-like activity. Here we show that both beta-C and CTX inhibit 3-methylcholanthrene (MCA)-induced transformation with ED50s of 9 x 10(-7) M and 2 x 10(-7) M, respectively. Both carotenoids failed to inhibit X-ray-induced transformation when the cells were treated prior to and during irradiation. However, when the drugs were added 1 week after X-irradiation and maintained in the medium thereafter, as in the chemical transformation protocol, both carotenoids inhibited subsequent development of transformed foci in a dose-dependent manner. Again, CTX was more effective than beta-C, such that 3 x 10(-6) M completely inhibited radiogenically-induced foci. Similar to the previously described action of retinoids, the inhibition of MCA-induced transformation was reversible; upon removal of the drug, transformed foci developed within 2 weeks, indicating that the carotenoids were not specifically toxic to initiated cells. Although both carotenoids caused a small dose-dependent decrease in the growth rate of both parental and initiated 10T1/2 cells, they did not markedly affect colony size or number when the cells were treated as in the transformation assays, nor did they influence the expression of neoplasia of two transformed cell lines. Although the actions of beta-C and CTX are similar to those of retinoids in the 10T1/2 system, we suggest that the carotenoids act via a different mechanism, since CTX cannot be converted to active retinoids in mammalian cells, and there is no evidence that 10T1/2 cells can convert beta-C to vitamin A. We suggest that the carotenoids' lipid anti-oxidant properties may be responsible for their inhibitory actions on transformation.

Suppression of large intestinal cancer in F344 rats by inositol hexaphosphate.

Abstract: Epidemiological data demonstrate correlations between dietary factors and the incidence of large intestinal cancer (LIC). Certain high-fiber diets are associated with a lower risk of LIC; these high-fiber diets are also rich in inositol hexaphosphate (IP6 or phytic acid). In a pilot study, we have used F344 rats to investigate the effect of sodium inositol hexaphosphate (Na-IP6) prior to (experiment I) and following injections of the carcinogen azoxymethane (AOM) (experiment II). In experiment I, rats started on 1% Na-IP6 in drinking water 1 week prior to the carcinogen treatment showed a 34.7% decrease (P less than 0.01) in LIC compared to control carcinogen treatment group. A similar reduction in the incidence of LIC was also observed in experiment II, wherein Na-IP6 supplementation was started 2 weeks following the last dose of the carcinogen. Comparison of the incidence of mitosis in the colonic crypts of the animals in different groups show that animals on AOM + IP6 demonstrate a significantly lower (P less than 0.001) mitotic rate than those receiving AOM only. Pilot studies of free radical generation demonstrate a reduction in .OH radical formation by Na-IP6. Further studies to expand this pilot data and to understand the mechanism of IP6 mediated LIC suppression are needed for it may have significance in our strategies for LIC control.

Use of ascorbic acid to inhibit nitrosation: kinetic and mass transfer considerations for an in vitro system.

Abstract: Ascorbic acid and ascorbate ion (denoted together as ASC) inhibit nitrosation by competing for the nitrosating agents formed from nitrite (e.g. N2O3, NO+ and NOSCN). ASC is oxidized irreversibly by this reaction and the nitrite equivalents are reduced to nitric oxide (NO). In open, aerobic systems the effective stoichiometry of the reaction between ASC and nitrite is not fixed, but is determined by a competition between the physical removal of NO (and NO2) from the system and the oxidation of NO by dissolved O2. The oxidation of NO reconverts it to a nitrosating agent which may react again with the remaining ASC. To determine the conditions under which ASC is most effective as a nitrosation inhibitor, we examined the kinetics of the reactions between nitrite and ASC and between nitrite and proline. These reactions were studied in open shaker flasks as functions of pH, anion composition (SCN- and Cl-), temperature, and gas-liquid mass transfer rate. At lower mass transfer rates, at lower pH and/or in the presence of SCN- or Cl-, relatively more ASC was consumed by a given amount of nitrite. Increased temperature caused more or less ASC to be consumed by a given amount of nitrite, depending on the conditions. A mathematical model of the reactions and mass transfer steps was developed which describes each of these features. The model predicts the variable stoichiometry of the reaction between nitrite and ASC in open, aerobic systems, and clarifies the mechanisms by which ASC inhibits nitrosation.

Transformation of rat liver epithelial cells with v-H-ras or v-raf causes expression of MDR-1, glutathione-S-transferase-P and increased resistance to cytotoxic chemicals.

Abstract: We have examined the relationship between transformation and multidrug resistance by employing the v-H-ras or v-raf oncogenes to transform rat liver epithelial (RLE) cells in vitro. The data indicate that transformation of RLE cells with v-H-ras or v-raf results in increased resistance to the cytotoxins adriamycin, vinblastine and 2-acetylaminofluorene. This multidrug resistance is accompanied by increasing expression of P-glycoprotein (MDR-1) and glutathione-S-transferase P (GST-P). Thus, neoplastic transformation of RLE cells with v-raf or v-H-ras, independently of chemical exposure, results in multidrug resistance.

Arteriosclerotic plaque development is 'promoted' by polynuclear aromatic hydrocarbons.

Abstract: In previous work we found that weekly injections of the polynuclear aromatic hydrocarbon (PAH) carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) induced spontaneous aortic plaques in cockerels to grow to a larger size and at a faster rate than plaques in control animals. To determine whether plaque-stimulating ability is related to carcinogenic potency or mutagenicity we have now tested a variety of agents, including PAH carcinogens, non-PAH carcinogens and weakly carcinogenic PAHs. Cockerels were injected weekly (from 4-20 weeks of age) with one of the following compounds: benzo[a]pyrene (B[a]P), benzo[e]pyrene (B[e]P), dibenz[a,h]anthracene (AH), dibenz[a,c]anthracene (AC), 3-methylcholanthrene (MCA), acetylaminofluorene (AAF), N-methyl-N,N'-nitro-nitrosoguanidine (MNNG) or anthracene (ANT). Plaques were present in the abdominal aortas of all animals. Plaque volumes were 8-14 times greater in AC-, B[a]P-, B[e]P-, MCA- and AH-treated cockerels than in controls. Plaques were slightly larger in the AAF-treated group than in control animals, and in the ANT- and MNNG-treated groups were indistinguishable in size from plaques in control animals. The largest plaque volumes were in AH-treated cockerels and were comparable in size to those elicited by DMBA treatment. The accelerated development of plaques is consistent with a 'promotional' role for these agents. There was a poor correlation between mutagenicity or carcinogenicity and plaque 'promotion', which may reflect a role for different metabolites in these processes.

Identification of multiple regulatory elements on the human cytochrome P450IA1 gene

Abstract: To examine the transcriptional regulation of the human cytochrome P450IA1 gene, a 3574 bp fragment containing 1140 bp of 5' flanking sequences, exon 1 (leader information only), intron 1, and the leader sequences from exon 2, was cloned upstream of the reporter gene, chloramphenicol acetyltransferase, and used to transfect the human hepatoma cell line, HepG2. In transient expression assays, treatment of the transfected cells with 3-methylcholanthrene, benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzofuran was shown to induce the expression of chloramphenicol acetyltransferase 10-fold. Previous studies by other investigators have identified a xenobiotic responsive element at greater than 800 bp 5' to the cap site in the mouse and rat cytochrome P450IA1 gene. In the current report, deletion of sequences from the 5' side of the P450IA1 fragment, as well as internal deletions, were used to identify at least three additional regulatory elements. A second positive, 3-methylcholanthrene responsive element was localized to sequences between -49 and -560 in addition to confirming the location of a similar element between -831 and -1140. These elements flank a potent negative regulatory element that has been conserved between the rat, mouse and human P450IA1 genes and also exhibits significant sequence identity with one of the negative control elements of the human c-Ha-ras1 proto-oncogene. Deletion of the negative control element clearly demonstrated that the fragments containing xenobiotic responsive elements also possess positive, constitutive control activity. A fourth element located within intron 1 was shown to potentiate the activity of 3-methylcholanthrene when the cells were treated simultaneously with the glucocorticoid agonist, dexamethasone.

Stable phenotypic expression of glutathione S-transferase placental type and unstable phenotypic expression of γ-glutamyltransferase in rat liver preneoplastic and neoplastic lesions

Abstract: Using immunohistochemical demonstration of glutathione S-transferase placental type (GST-P) and histochemical demonstration of gamma-glutamyltransferase (gamma-GT), the long-term development of preneoplastic and neoplastic lesions was followed in rats over a 50-week period. Rats were given a single i.p. injection of 200 mg/kg body weight of diethylnitrosamine (DEN), and then 2 weeks later were administered 0.02% 2-acetylaminofluorene (2-AAF) (group 1), 0.05% phenobarbital (PB) (group 2), 2.0% butylated hydroxyanisole (BHA) (group 3) or no supplement (group 4) in their diet for 6 weeks, all rats being subjected to partial hepatectomy at week 3. Hepatocellular proliferated lesions were classified as foci, nodules and hepatocellular carcinomas. Development of foci, nodules and hepatocellular carcinomas was enhanced strongly by 2-AAF and weakly by PB, and inhibited by BHA. Almost all foci and nodules were GST-P positive, although 5-10% of the GST-P-positive foci were gamma-GT negative. The areas of GST-P-positive foci and nodules increased with time in all groups. In contrast, while the areas of gamma-GT-positive lesions also increased with time in groups 2-4, they decreased from week 12 in group 1. As the percentage gamma-GT-positive area in GST-P-positive foci significantly decreased with time in all groups, the rate of phenotypic reversion of gamma-GT in foci in group 1 was revealed to be larger than the focus growing rate, whereas that in groups 2-4 was smaller. Gamma-GT-negative and GST-P-positive micro-nodules of altered morphology appeared within gamma-GT- and GST-P-positive nodules in later stages. All hepatocellular carcinomas found in this experiment consisted of GST-P-positive cells. In contrast, 37% (13/35) of the hepatocellular carcinomas were negative for gamma-GT. The results indicate GST-P to be the most accurate marker enzyme for detection of initiated cells during liver carcinogenesis and gamma-GT to be more appropriate for indicating changes of phenotypic expression in each lesion type.

Active oxygen induced DNA strand breakage and poly ADP-ribosylation in promotable and non-promotable JB6 mouse epidermal cells.

Abstract: The evidence is convincing that oxidants and agents which induce a cellular pro-oxidant state can act as carcinogens, in particular as promoters and progressors. Importantly, infiltrated phagocytes represent a source of oxidants in inflamed tissues. We have studied the mechanism of the promotional action of active oxygen (AO) in mouse epidermal cells JB6 by comparing the non-promotable clone 30 to the promotable clone 41. In order to mimick AO released by phagocytes we used xanthine/xanthine oxidase as a source of extracellular superoxide and hydrogen peroxide. We found that AO stimulated the growth only of promotable clone 41 after an initial period of moderate inhibition while it was strongly cytostatic for non-promotable clone 30. Reasons for the higher cytostatic effect of AO on the non-promotable clone 30 were discovered when we measured DNA strand breakage and poly ADP-ribosylation of chromosomal proteins. At equal doses AO induced 4-5 times more DNA breaks in clone 30 in reactions which required iron--and probably also calcium--ions. The higher amount of DNA breakage in clone 30 was reflected in a higher extent of poly ADP-ribosylation. Excessive DNA breakage and poly ADP-ribosylation which causes the depletion of NAD and ATP may be responsible for the strong cytostatic effect of AO in clone 30. We conclude that differential resistance to the cytostatic/cytotoxic effect of AO in part determines the promotability of mouse epidermal cells JB6.

The impact of toxicity on carcinogenicity studies: implications for risk assessment

Abstract: This paper explores the inter-relationship between toxicity, genotoxicity, and carcinogenicity in laboratory rodents. To our knowledge this is the first attempt to integrate these factors and evaluate their implications for the process of risk assessment. The evaluation is based on information obtained from 2-year laboratory-animal studies involving 99 chemicals. The data suggest that only seven of the 53 positive carcinogenicity studies exhibited the types of target organ toxicity that could have been the cause of all observed carcinogenic effects. Furthermore, no apparent difference in mutagenicity as measured by the Ames Salmonella assay was observed between 'high dose only' carcinogens and the entire set of carcinogens. These findings suggest that the number of chemical carcinogens that we can identify solely through rodent studies as being potential tumor inducers through some indirect mechanism is small. Generally speaking, the identification of histopathological effects is not sufficient in itself for justifying mechanistic assumptions, and supplemental biological information will be necessary to reach definitive conclusions.

Genetic toxicology of lead compounds

Abstract: We have investigated the activity of insoluble and soluble lead compounds in inducing mutagenesis, cell transformation and sister chromatid exchange in mammalian cells. Insoluble lead sulfide, readily phagocytized, was more than four times as toxic to V79 cells on a microM basis, than two moderately soluble lead compounds although the exposure time for the soluble salts was five times longer. These findings demonstrate the importance of different cellular mechanism(s) of metal uptake and bioavailability. Both insoluble lead sulfide and more soluble lead nitrate were mutagenic at the HPRT locus in V79 cells. Although less mutagenic at the higher concentrations, lead nitrate at a concentration of 500 microM enhanced the mutation frequency greater than 6-fold above background following a 5-day exposure. Although the mechanism(s) by which lead induces mutations is unknown, failure of both compounds to induce SCE and DNA single-strand breaks, detectable by alkaline elution, suggests that lead-induced mutations may not be a result of direct damage to DNA but may occur via indirect mechanisms including disturbances in enzyme functions important in DNA synthesis and/or repair, or in DNA-helical structure. Lead acetate also transformed SHE cells in a dose-response fashion following a 48-h exposure. Our results indicate that lead compounds may be genotoxic by an indirect mechanism, and lend support to the view that lead is a carcinogen.

Evidence for 4-(3-pyridyl)-4-oxobutylation of DNA in F344 rats treated with the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine.

Abstract: DNA was isolated from tissues of F344 rats 24 h after treatment by s.c. injection with [5-3H]4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNK) or [5-3H]N'-nitrosonornicotine ([5-3H]NNN). It was hydrolyzed with acid or at pH 7, 100 degrees C, and the hydrolysates were analyzed by HPLC. The major product in each case was identified as 4-hydroxy-1-(3-pyridyl)-1-butanone, formed by hydrolysis of a DNA adduct. It was detected in DNA from the livers of rats treated with [5-3H]NNK or [5-3H]NNN, and in DNA from lungs of rats treated with [5-3H]NNK. These results demonstrate that 4-(3-pyridyl)-4-oxobutylation of DNA occurs in rats treated with NNK or NNN, and are consistent with the hypothesis that these nitrosamines are metabolically activated by alpha-hydroxylation.

Detection of activated proto-oncogenes in N-nitrosodiethylamine-induced liver tumors: a comparison between B6C3F1 mice and Fischer 344 rats.

Abstract: DNA from B6C3F1 mouse and Fischer 344 rat liver tumors induced by N-nitrosodiethylamine (DEN) were examined for the ability to induce morphological transformation of NIH3T3 cells. DNAs from 14 of 33 of the mouse liver tumors induced by a single injection of DEN at 12 or 15 days of age were positive in this assay while DNA from only one of 28 DEN-induced rat liver tumors was active. Southern blot analysis of the NIH3T3 transformants derived from the mouse liver tumors revealed amplified and/or rearranged restriction fragments homologous to the H-ras proto-oncogene. DNA from two independent foci induced by the rat tumor DNA did not hybridize to probes for members of the ras gene family or c-raf. Activating mutations in the H-ras genes from the DEN-induced mouse liver tumors were characterized by selective oligonucleotide hybridization and the detection of a new XbaI restriction site by Southern blot analysis. In activated H-ras genes from the DEN-induced mouse liver tumor DNA, seven of 14 had a CG----AT transversion at the first base of the 61st codon, three of 14 had an AT----GC transition and four of 14 had the AT----TA transversion at the second base of codon 61. This spectrum of mutations is very similar to that recently observed in activated H-ras genes found in spontaneously occurring B6C3F1 mouse liver tumors. Taken together, the data suggest that the DEN-induced rat and mouse liver carcinogenesis may involve genetic targets other than or in addition to the H-ras gene.

c-H-ras and c-K-ras gene hypomethylation in the livers and hepatomas of rats fed methyl-deficient, amino acid-defined diets.

Abstract: The extent of methylation of the c-H-ras and c-K-ras oncogenes was compared in neoplastic and preneoplastic livers of rats fed one of several methyl-deficient, amino acid-defined diets for 18 months, with or without a preceding initiating dose of diethylnitrosamine (DEN). The restriction endonucleases MspI, HpaII, HhaI, PaeR71 and XhoI were used for studying the extent and pattern of DNA methylation. The results indicated that both c-H-ras and c-K-ras oncogenes were hypomethylated in all DNA samples derived from both neoplastic and preneoplastic livers of rats fed any of the methyl-deficient diets used, regardless of whether or not the rats had received an initiating dose of DEN. It thus appears that dietary methyl deficiency does indeed lead to hypomethylation of ras genes in the DNAs of the resulting tumors. However, the significance of this hypomethylation in the tumorigenic process is not clearly understood.

Liver cell proliferation and incidence of hepatocellular carcinomas in rats fed consecutively a choline-devoid and a choline-supplemented diet.

Abstract: A group-set of male Fischer 344 rats was kept on a choline-devoid (CD) diet for 3, 6, 9, 12 or 16 months. A second set was fed the same diet for 3, 6 and 9 months, followed by a control, choline-supplemented diet for 3 months. A third set was fed the CD diet for 0, 3, 6, 9 and 12 months and the control diet for the duration (16 months) of the experiment. [3H]Thymidine was injected into some of the animals to assess the extent of liver cell proliferation. In the latter animals, liver triacylglycerols were also determined as an index of the hepatonecrogenic action of a CD diet. Foci of enzyme-altered hepatocytes were detected histochemically and the presence of tumors was established histologically. Cell proliferation and triacylglycerols were both high after 3 months and declined steadily as the length of CD diet feeding increased. Upon subsequent feeding with the control diet, triacylglycerols promptly cleared from the liver, while cell proliferation remained at reduced but still relatively high levels for at least 3 months. Increasing but small numbers of foci of enzyme-altered hepatocytes were detected in some of the rats under experimentation for 6 months or longer. The incidence of hepatocellular carcinomas was 13, 27, 33 and 73% respectively in rats fed the CD diet for 3, 6, 9 or 12 months and the control diet for the duration of the experiment. On the other hand, only 26% of the rats fed exclusively the CD diet for 16 months developed carcinomas. The results were taken as evidence that: (i) liver cell proliferation persists beyond discontinuation of a CD diet; (ii) the liver contains initiated cells--capable of full evolution to cancer even in the absence of active promotion--after a relatively short 3-month exposure to a CD diet; and (iii) occurrence of a late event(s) is critical for the genesis of the tumors. Whether a CD diet is a complete carcinogen able to initiate de novo liver cells or acts merely as a promoter of the evolution of endogenous initiated cells is briefly discussed.

Effects of dietary menhaden oil and retinyl acetate on the growth of DU 145 human prostatic adenocarcinoma cells transplanted into athymic nude mice.

Abstract: The effects of feeding menhaden oil (MO), rich in omega-3 fatty acids, or supplemental vitamin A [as retinyl acetate (RA)], on the growth of DU 145 human prostate cancer cells were studied in athymic nude mice. The mice were fed AIN-76A diets containing either 23% corn oil (CO), a mixture of 17% MO and 6% CO, or 23% CO plus RA. After irradiation sterilization, the RA-supplemented diet was found to contain approximately 15 times the amount of vitamin A present in the control diet. There were 24 mice in each dietary group. Three weeks after commencement of feeding the experimental diets, 1 x 10(6) or 5 x 10(6) DU 145 cells were inoculated into subgroups of 12 animals, and the appearance and growth of solid tumors followed over a 6-week period. There was no significant difference in tumor latency between mice fed MO plus CO, and those fed CO alone, regardless of the inoculum size. However, the appearance of palpable tumors was more rapid in mice inoculated with 5 x 10(6) cells and fed the RA-supplemented CO diet (91% after 17 days) compared with mice receiving the same tumor cell load but fed the unsupplemented CO diet (55% after 17 days). Growth of the solid tumors was retarded significantly in mice inoculated with 1 x 10(6) cells and fed the MO-containing diet compared with the CO controls; this effect was not evident in animals who received 5 x 10(6) cells. RA supplementation caused accelerated tumor growth, which, again, only achieved statistical significance in the group inoculated with 1 x 10(6) cells.

Repair of alkylated purines in the hepatic DNA of mitochondria and nuclei in the rat

Abstract: Further evidence for the preferential interaction of carcinogens with mitochondrial DNA (mtDNA) has been obtained. In rats treated with high doses of N-nitrosodimethylamine (NDMA) or N-nitroso-N-butylurea (NBU), hepatic mtDNA contains 1.4 times more O6-methyl-2'-deoxyguanosine (O6-MedG) or 2.3 times more O6-butyl-2'-deoxyguanosine (O6-BudG) than does nuclear DNA (nDNA). The kinetics of removal of O6-MeG from mtDNA and nDNA are similar at both high (20 mg/kg) and low (2 mg/kg) doses of NDMA, and the removal of O6-MeG can be increased by pretreating the animals with 2-acetylaminofluorene (AAF), indicating that O6-MeG is repaired in the mitochondrion by a mechanism similar to that which functions in the nucleus. In contrast, O6-BudG is removed very slowly from mtDNA and rapidly from nDNA, an observation which is consistent with the absence of a nucleotide excision mechanism in the mitochondrion and the repair of O6-BudG, predominantly by an excision mechanism, in the nucleus of mammalian cells. A 23-kd methyltransferase (MT) protein, similar to the one found in the nucleus, has been isolated from hepatic mitochondria and is present in mitochondria from which the outer membrane has been removed. It is suggested that O6-MeG, but not O6-BuG can be repaired from mtDNA by a MT protein that is nuclear encoded and transported across the mitochondrial membrane.

The measurement of cisplatin-DNA adduct levels in testicular cancer patients.

Abstract: Seventeen patients with 'poor prognosis' non-seminomatous testicular cancer were monitored for formation of intrastrand bidentate N7-d(ApG)- and N7-d(GpG)-diammineplatinum adducts in peripheral blood cell DNA during the course of cisplatin-based chemotherapy. Adduct values from blood cell DNA samples were compared with disease response data from the same individuals. Patients who received a dose of 40 mg/m2 cisplatin for 5 days generally formed more adducts than patients receiving 20 mg/m2 for 5 days, and adduct levels ranged from 0 to approximately 300 amol/micrograms DNA. Among the individuals who achieved a complete response, the median adduct level was 170 amol/micrograms DNA and the mean was 162. Among the individuals who achieved a partial response, the median adduct level was 78 amol/micrograms DNA and the mean was 83. Comparison of adduct levels between response groups using the Mann-Whitney test gave a two-sided P value of 0.072 (one-sided P value 0.036). Of 11 patients forming high levels of adduct (greater than 140 amol/micrograms DNA), 10 achieved a complete response; this compares with two complete responders in the group of six patients forming low levels (less than 100 amol/micrograms DNA) of adduct (P = 0.055, two-sided Fisher exact test). We conclude that cisplatin-DNA adduct formation in peripheral blood cell DNA correlates with the occurrence of complete response in patients with poor prognosis testicular cancer.