Different genetic alterations in rat forestomach tumors induced by genotoxic and non-genotoxic carcinogens. (25/170)

Human beings are exposed to a multitude of carcinogens in their environment, and most cancers are considered to be chemically induced. Here we examined differences in genetic alterations in rat forestomach tumors induced by repeated exposure to a genotoxic carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or N-methylnitrosourethane (MNUR), and chronic treatment with a non-genotoxic carcinogen, butylated hydroxyanisole (BHA) or caffeic acid (CA). A total of 132, 6-week-old male F344 rats were employed. Forty rats were treated with MNNG by intragastric administration at a dose of 20 mg/kg body wt once a week for 32 weeks, and 20 rats received 20 p.p.m. MNUR in their drinking water for 48 weeks. Further groups of 20 animals were administered 2% BHA or 2% CA in the diet for 104 weeks. The remaining rats were maintained without any supplement as controls. Multiple forestomach tumors were observed in all rats of the MNNG-, MNUR-, BHA- and CA-treated groups. Histopathologically, MNUR- and CA-treated groups showed almost the same pattern. On polymerase chain reaction-single strand conformation polymorphism analysis, H-ras and p53 gene mutations were observed at high and relatively low frequencies, respectively, in forestomach tumors induced by MNNG and MNUR. Most H-ras gene mutations were G-->A transitions in codons 7 and 12 of exon 1. On the other hand, forestomach tumors due to the non-genotoxic carcinogens, BHA and CA, had almost no mutations of the H-ras and p53 genes. Moreover, relative overexpression of cyclin D1 and p53 was detected in forestomach tumors induced by the genotoxic carcinogens, while their non-genotoxic counterparts had a tendency to show low expression of those molecules. Mutations of the beta-catenin gene were not detected in any group. The present study demonstrates that rat forestomach tumors induced by genotoxic and non-genotoxic carcinogens have different underlying genetic alterations, even if their pathological features are similar.  (+info)

The as-1 promoter element is an oxidative stress-responsive element and salicylic acid activates it via oxidative species. (26/170)

The activation sequence-1 (as-1)-like element found in the promoter of some glutathione S-transferase (GST) genes, has been previously described as a salicylic acid (SA)- and auxin-responsive element. In this paper, we tested the hypothesis that the activating effect of SA on the as-1 element is mediated by oxidative species. Supporting this hypothesis, our results show that the antioxidants dimethylthiourea (DMTU) and 3-t-butyl-4-hydroxy-anizole (BHA) inhibit the SA-induced transcription of genes controlled by as-1 elements in tobacco (Nicotiana tabacum) plants [i.e. GNT35 gene coding for a GST and (as-1)(4)/beta-glucuronidase (GUS) reporter transgene]. DMTU and BHA also inhibit SA-activated as-1-binding activity in nuclear extracts. Further support for the hypothesis that the as-1 element is activated by oxidative species comes from our result showing that light potentiates the SA-induced activation of the as-1 element. Furthermore, methyl viologen, a known oxidative stress inducer in plants, also activates the as-1 element. Increasing H(2)O(2) levels by incubation with H(2)O(2) or with the catalase inhibitor 3-amino-1,2,5-triazole does not activate the (as-1)(4)/GUS gene. On the contrary, 3-amino-1,2,5-triazole inhibits the activating effect of SA on the (as-1)(4)/GUS gene. These results suggest that oxidative species other than H(2)O(2) mediate the activation of the as-1 element by SA. Our results also suggest that even though the as-1 binding activity is stimulated by oxidative species, this is not sufficient for the transactivation of genes controlled by this element. The complex interplay between SA and reactive oxygen species in the transcriptional activation of defense genes is discussed.  (+info)

Estimation of inorganic food additive (nitrite, nitrate and sulfur dioxide), antioxidant (BHA and BHT), processing agent (propylene glycol) and sweetener (sodium saccharin) concentrations in foods and their daily intake based on official inspection results in Japan in fiscal year 1998. (27/170)

The mean concentration and daily intake of inorganic food additives (nitrite, nitrate, and sulfur dioxide), antioxidants (BHA and BHT), a processing agent (propylene glycol), and a sweetener (sodium saccharin) were estimated based on the results of an analysis of 34,489 food samples obtained in official inspections by 106 local governments in Japan in fiscal year 1998. The ratios of mean concentrations of these seven food additives to each allowable limit were 20.0%, 53.9%, 15.5%, 6.2%, 0.4%, 18.5%, and 5.7%, respectively. The daily intakes of these food additives estimated from their concentrations in foods and the daily consumption of foods were 0.205, 0.532, 4.31, 0.119, 0.109, 77.5, and 7.27 mg per person, respectively. These amounts were 6.8%, 0.3%, 12.3%, 0.5%, 0.7%, 6.2%, and 2.6% of the acceptable daily intake (ADI), respectively, when body weight was assumed to be 50 kg. No remarkable differences in the daily intakes of these seven food additives or the ratios to the ADI were observed compared with the results based on the official inspections in fiscal years 1994 and 1996.  (+info)

Meat and cancer: haemoglobin and haemin in a low-calcium diet promote colorectal carcinogenesis at the aberrant crypt stage in rats. (28/170)

High intake of red meat, but not of white meat, is associated with an increased risk of colon cancer. However, red meat does not promote cancer in rodents. Haemin, added to low-calcium diets, increases colonic proliferation, and haemoglobin, added to high-fat diets, increases the colon tumour incidence in rats, an effect possibly due to peroxyl radicals. We thus speculated that haem might be the promoting agent in meat, and that prevention strategies could use calcium and antioxidants. These hypotheses were tested in rats at the aberrant crypt foci (ACF) stage at 100 days. F344 rats (n = 124) were given an injection of azoxymethane and were then randomized to 11 groups fed with low-calcium (20 micro mol/g) AIN76-based diets, containing 5% safflower oil. Haemin (0.25, 0.5 and 1.5 micro mol/g) or haemoglobin (1.5 and 3 micro mol haem/g) was added to five experimental diets, compared with a control diet without haem. Three other high-haemin diets (1.5 micro mol/g) were supplemented with calcium (250 micro mol/g), antioxidant butylated hydroxyanisole and rutin (0.05% each), and olive oil, which replaced safflower oil. Faecal water was assayed for lipid peroxidation by thiobarbituric acid reactive substances (TBARs) test, and for cytolytic activity. Haemin strikingly increased the ACF size, dose-dependently, from 2.6 to 11.4 crypts/ACF (all P < 0.001). The high-haemin diet also increased the number of ACF per colon (P < 0.001). Promotion was associated with increased faecal water TBARs and cytotoxicity. Calcium, olive oil and antioxidants each inhibited the haemin-induced ACF promotion, and normalized the faecal TBARs and cytotoxicity. The haemoglobin diets increased the number of ACF and faecal TBARs, but not the ACF size or the faecal cytotoxicity. In conclusion, dietary haemin is the most potent known ACF promoter. Haemoglobin is also a potent promoter of colorectal carcinogenesis. The results suggest that myoglobin in red meat could promote colon cancer. Diets high in calcium, or in oxidation-resistant fats, may prevent the possible cancer-promoting effect of red meat.  (+info)

Inhibition of preadipocyte proliferation by mitochondrial reactive oxygen species. (29/170)

Preadipocytes are present and can proliferate to increase fat mass throughout adult life. The importance of mitochondria in these cells has never been investigated, although we recently reported that mitochondrial oxidative metabolism is non-negligible in white preadipocytes. Mitochondrial reactive oxygen species generation is intimately associated with respiratory chain function. An increasing number of reports support their role as signalling molecules. The aim of this work was to study the effects of mitochondrial reactive oxygen species on proliferation of white preadipocytes. Rotenone and oligomycin, inhibitors of complex I and of ATP synthase respectively, increased H(2)O(2) and inhibited cell growth of preadipocytes (without inducing necrosis or apoptosis). These effects were partly prevented by addition of radical scavengers. A chemical uncoupler had opposite effects on reactive oxygen species generation and cell growth. Propofol, which inhibits complex I but also scavenges free radicals, had effects similar to those of the uncoupler on both parameters. Thus, mitochondrial reactive oxygen species can influence development of adipose tissue by affecting the size of the white preadipocyte pool.  (+info)

Regulation of human NAD(P)H:quinone oxidoreductase gene. Role of AP1 binding site contained within human antioxidant response element. (30/170)

Deletion mutagenesis and transfection studies into hepatic (mouse hepatoma (Hepa-1) and human hepatoblastoma (Hep-G2)) and nonhepatic (HeLa) cells indicated that high levels of expression of the human NAD(P)H:quinone oxidoreductase gene in tumor cells and its induction by beta-naphthoflavone and 3-(2)-tert-butyl-4-hydroxyanisole are mediated by human antioxidant response element (hARE) located in the region between -470 and -445. The hARE, when attached to the thymidine kinase promoter and transfected into several mammalian cells, expressed high levels of the chloramphenicol acetyltransferase gene that was inducible by beta-naphthoflavone and 3-(2)-tert-butyl-4-hydroxyanisole. Nucleotide sequence analysis of the hARE revealed the presence of a recognition site for binding to the AP1 protein. Mutation of the AP1 binding site located within the hARE resulted in the loss of expression and induction upon transfection into various cell types. Band shift and competition assays with hARE and nuclear extracts from control and beta-naphthoflavone-treated Hepa-1, Hep-G2 and HeLa cells indicated specific interaction of regulatory protein(s) to the hARE. The supershift assays using antibodies against specific proteins of the AP1 family identified Jun-D and c-Fos as two members in the hARE-protein complex observed in band shift assays.  (+info)

Nitroreductases and glutathione transferases that act on 4-nitroquinoline 1-oxide and their differential induction by butylated hydroxyanisole in mice. (31/170)

These studies concern the initial steps in 4-nitroquinoline 1-oxide (4NQO) metabolism in relation to mechanisms of anticarcinogenesis. Butylated hydroxyanisole (BHA) administration by a protocol known to inhibit the pulmonary tumorigenicity of 4NQO in A/HeJ mice enhanced hepatic and pulmonary activities for 4NQO metabolism by two major pathways, conjugative detoxification and nitroreductive activation. High-performance liquid chromatography analysis showed approximate doubling of two types of glutathione transferase subunits with 4NQO-conjugating activity in livers of BHA-treated mice. Similar increases were observed in hepatic 4NQO-conjugating activity and in Vmax, while Km for 4NQO was 39 to 43 microM. Pulmonary 4NQO-glutathione transferase activity increased 24 to 29%. DT diaphorase activity toward 4NQO was elevated 3.3-fold in livers and 2.7-fold in lungs of BHA-treated mice. However, the predominant 4NQO reductase of liver and lung was dicumarol resistant, had a strong preference for NADH, and showed little if any response to BHA. This Mr 200,000 enzyme, partially purified from livers of Swiss mice, exhibited the stoichiometry of 2-NADH/4NQO expected for reduction of 4NQO to 4-hydroxyaminoquinoline 1-oxide. Its high affinity for 4NQO (Km, 15 microM) signified a much greater influence on 4NQO metabolism than DT diaphorase (Km, 208 microM). The dicumarol-resistant 4NQO reductase differed from several known cytosolic nitroreductases. The results suggest that protection by BHA may result from alteration of the balance between 4NQO activation and conjugation.  (+info)

Photoaffinity labelling of steroid-hormone-binding glutathione S-transferases with [3H]methyltrienolone. Inhibition of steroid-binding activity by the anticarcinogen indole-3-carbinol. (32/170)

The identification and characterization of steroid-hormone-binding glutathione S-transferases (GST) were undertaken using photoaffinity-labelling techniques. Irradiation of mouse liver cytosol, in the presence of 50 nM-[3H]methyltrienolone, resulted in the specific affinity labelling of five proteins. One of these proteins, designated MBP27, had an approximate molecular mass of 27 kDa under denaturing conditions and was induced by treatment of mice with either 2(3)-t-butyl-4-hydroxyanisole (BHA) or phenobarbital (PB). An additional affinity-labelled protein, MBP25, which was not detected in untreated mouse cytosol, was induced in the liver cytosols from BHA- and PB-treated mice. The molecular masses of these proteins and their induction by BHA and PB suggested that they may be steroid-hormone-binding GST subunits. Irradiation of mouse liver cytosol in the presence of [3H]methyltrienolone, followed by immunoprecipitation using GST-specific antibodies established that both GST mu and GST alpha bind [3H]methyltrienolone and both contribute to the affinity-labelled protein designated MBP27. GST Ya1 Ya1, an alpha class GST that is not expressed in untreated mouse liver but is induced by BHA and PB, was also found to bind [3H]methyltrienolone and is identical with the affinity-labelled protein designated MBP25. Experiments were undertaken next to assess the effects of the anticarcinogenic plant compound indole-3-carbinol (I3C) on GST-mediated steroid hormone-binding using the photoaffinity labelling techniques. Treatment of mice with I3C resulted in the induction of immunoreactive GST mu and GST Ya1 Ya1. However, the steroid-binding activity of these proteins in vitro was severely inhibited by the acid-condensation products of I3C that are generated in the stomach after ingestion. These results suggest that I3C may inhibit GST-mediated steroid-binding activity which could contribute to the anticarcinogenic activity of this compound.  (+info)