Rapid characterization and pharmacokinetic study of aristolochic acid analogues using ion mobility mass spectrometry.

Aristolochic acid analogues (AAAs), naturally existing in herbal Aristolochia and Asarum genera, were once widely used in traditional pharmacopeias because of their anti-inflammatory properties, but lately they were identified as potential nephrotoxins and mutagens. A method for rapid characterization of AAAs in serum was developed using ion mobility spectrometry coupled with mass spectrometry (IMS-MS). Five AAAs, containing four aristolochic acids and one aristolactam, were separated and identified within milliseconds. AAAs were separated in gas phase based on the difference of their ion mobility (K0), and then identified based on their K0 values, m/z, and product ions from MS/MS. Quantitative analysis of AAAs was performed using an internal standard with a satisfactory sensitivity. Limits of detection (signal-to-noise = 3) and quantification (signal-to-noise = 10) were 1-5 ng/mL and 3-8 ng/mL, respectively. The method was validated and successfully applied to the pharmacokinetics study of AAAs in rats, offering a promising way for fast screening and evaluation of AAAs in biological samples.

Genotoxicity of heterocyclic amines (HCAs) on freshly isolated human peripheral blood mononuclear cells (PBMC) and prevention by phenolic extracts derived from olive, olive oil and olive leaves.

In this study we investigated the genotoxic potential of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, (PhIP); 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline, (IQ); 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline, (MeIQx) and 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (DiMeIQx) on human freshly isolated peripheral blood mononuclear cells (PBMC) by the comet assay. The preventive ability of three different phenolic extracts derived from olive (O-PE), virgin olive oil (OO-PE) and olive leaf (OL-PE) on PhIP induced DNA damage was also investigated. PhIP and IQ induced a significant DNA damage at the lowest concentration tested (100 µM), while the genotoxic effect of MeIQx and DiMeIQx become apparent only in the presence of DNA repair inhibitors Cytosine b-D-arabinofuranoside and Hydroxyurea (AraC/HU). The inclusion of metabolic activation (S9-mix) in the culture medium increased the genotoxicity of all HCAs tested. All three phenolic extracts showed an evident DNA damage preventive activity in a very low concentration range (0.1-1.0 µM of phenols) which could be easily reached in human tissues "in vivo" under a regular intake of virgin olive oil. These data further support the observation that consumption of olive and virgin olive oil may prevent the initiation step of carcinogenesis. The leaf waste could be an economic and simple source of phenolic compounds to be used as food additives or supplements.

Investigations into the genotoxic potential of olive extracts.

The phenolic anti-oxidant 3-hydroxytyrosol (HT) is a major constituent of olives and olive oil. Published data showed it was negative in the Ames test at concentrations up to 5 µL per plate, but did induce chromosomal aberrations in human lymphocytes. HIDROX, an olive extract containing approximately 2.4% HT, was reported as both positive and equivocal in an Ames test in different papers from the same laboratory. Negative results for micronucleus induction in vivo in both an acute study and as part of a 90-day rat toxicity study were also reported for HIDROX. Given the widespread use and consumption of olives, olive oil and olive extracts, it was important to obtain more data. Here we confirm that pure HT, and an olive extract containing 15% HT, both induced micronuclei in cultured cells in vitro, but show that these responses were either due to high levels of cytotoxicity or to reaction of HT with culture medium components to produce hydrogen peroxide. Another extract (H40) containing 40% HT also induced micronuclei in vitro, probably via the same mechanism. However, both extracts were negative in robust Ames tests. The 15% HT formulated extract did not induce micronuclei in rat bone marrow after 4 weeks of dosing up to 561 mg HT/kg/day. H40 produced increased rat bone marrow micronucleus frequencies at 250 and 500 mg HT/kg/day in a 90-day toxicity study, but the results were questionable for various reasons. However, when two different batches of this extract were tested in acute micronucleus studies at doses up to 2000 mg HT/kg, giving plasma exposures that exceeded those in the 90-day study, negative results were obtained. Based on weight of evidence it is concluded that the olive extracts tested are not genotoxic at high doses in vivo, and any genotoxic risks for human consumers are negligible.

Exposure-dose-response of Tellina deltoidalis to contaminated estuarine sediments 3. Selenium spiked sediments.

The metalloid selenium is an essential element which at slightly elevated concentrations is toxic and mutagenic. In Australia the burning of coal for power generation releases selenium into estuarine environments where it accumulates in sediments. The relationship between selenium exposure, dose and response was investigated in the deposit feeding, benthic, marine bivalve Tellina deltoidalis. Bivalves were exposed in microcosms for 28 days to individual selenium spiked sediments, 0, 5 and 20 µg/g dry mass. T. deltoidalis accumulated selenium from spiked sediment but not in proportion to the sediment selenium concentrations. The majority of recovered subcellular selenium was associated with the nuclei and cellular debris fraction, probably as protein bound selenium associated with plasma and selenium bound directly to cell walls. Selenium exposed organisms had increased biologically detoxified selenium burdens which were associated with both granule and metallothionein like protein fractions, indicating selenium detoxification. Half of the biologically active selenium was associated with the mitochondrial fraction with up to 4 fold increases in selenium in exposed organisms. Selenium exposed T. deltoidalis had significantly reduced GSH:GSSG ratios indicating a build-up of oxidised glutathione. Total antioxidant capacity of selenium exposed T. deltoidalis was significantly reduced which corresponded with increased lipid peroxidation, lysosomal destabilisation and micronuclei frequency. Clear exposure-dose-response relationships have been demonstrated for T. deltoidalis exposed to selenium spiked sediments, supporting its suitability for use in selenium toxicity tests using sub-lethal endpoints.

Arsenic and selenium toxicity and their interactive effects in humans.

Arsenic (As) and selenium (Se) are unusual metalloids as they both induce and cure cancer. They both cause carcinogenesis, pathology, cytotoxicity, and genotoxicity in humans, with reactive oxygen species playing an important role. While As induces adverse effects by decreasing DNA methylation and affecting protein 53 expression, Se induces adverse effects by modifying thioredoxin reductase. However, they can react with glutathione and S-adenosylmethionine by forming an As-Se complex, which can be secreted extracellularly. We hypothesize that there are two types of interactions between As and Se. At low concentration, Se can decrease As toxicity via excretion of As-Se compound [(GS3)2AsSe](-), but at high concentration, excessive Se can enhance As toxicity by reacting with S-adenosylmethionine and glutathione, and modifying the structure and activity of arsenite methyltransferase. This review is to summarize their toxicity mechanisms and the interaction between As and Se toxicity, and to provide suggestions for future investigations.

Toxicological assessment of 3-chloropropane-1,2-diol and glycidol fatty acid esters in food.

Fatty acid esters of 3-chloropropane-1,2-diol (3-MCPD) and glycidol are a newly identified class of food process contaminants. They are widespread in refined vegetable oils and fats and have been detected in vegetable fat-containing products, including infant formulas. There are no toxicological data available yet on the 3-MCPD and glycidol esters, and the primary toxicological concern is based on the potential release of 3-MCPD or glycidol from the parent esters by lipase-catalyzed hydrolysis in the gastrointestinal tract. Although 3-MCPD is assessed as a nongenotoxic carcinogen with a tolerable daily intake (TDI) of 2 µg/kg body weight (bw), glycidol is a known genotoxic carcinogen, which induces tumors in numerous organs of rodents. The initial exposure estimates, conducted by Federal Institute for Risk Assessment (BfR) under the assumption that 100% of the 3-MPCD and glycidol are released from their esters, revealed especially that infants being fed commercial infant formula could ingest harmful amounts of 3-MCPD and glycidol. However, the real oral bioavailability may be lower. As this gives rise for toxicological concern, the currently available toxicological data of 3-MCPD and glycidol and their esters are summarized in this review and discussed with regard to data gaps and further research needs.

Apiaceous vegetable constituents inhibit human cytochrome P-450 1A2 (hCYP1A2) activity and hCYP1A2-mediated mutagenicity of aflatoxin B1.

In humans, apiaceous vegetables (carrots, parsnips, celery, parsley, etc.) inhibit cytochrome P-450 1A2, a biotransformation enzyme known to activate several procarcinogens, including aflatoxin B1 (AFB). We evaluated eight phytochemicals from apiaceous vegetables for effects on human cytochrome P-450 1A2 (hCYP1A2) activity using a methoxyresorufin O-demethylase (MROD) assay and a trp-recombination assay. Saccharomyces cerevisiae was used for heterologous CYP1A2 expression and this yeast strain is also diploid and auxotrophic for tryptophan due to mutations in the trp5 alleles. When these two alleles undergo AFB-induced mitotic recombination, gene conversion occurs, allowing yeast to grow in the absence of tryptophan. The apiaceous constituents psoralen, 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), and apigenin were potent inhibitors of hCYP1A2-mediated MROD activity in yeast microsomes, whereas quercetin was a modest hCYP1A2 inhibitor. Naringenin, caffeic acid, and chlorogenic acid did not inhibit hCYP1A2-mediated MROD activity. The 2-h pretreatment of intact yeast cells with psoralen, 5-MOP, and 8-MOP significantly improved cell survival after subsequent 4-h AFB treatment and reduced hCYP1A2-mediated mutagenicity of AFB. Apigenin also significantly decreased mutagenicity. These results suggest that in vivo CYP1A2 inhibition by apiaceous vegetables may be due to the phytochemicals present and imply that apiaceous vegetable intake may be chemopreventive by inhibiting CYP1A2-mediated carcinogen activation.

Effect of black tea intake on the excretion of mutagens in the urine of volunteers taking a beef meal.

The objective of this study was to investigate in a crossover study conducted in human volunteers whether black tea intake modulates the metabolism of heterocyclic amines, consumed in the form of well-cooked beefburgers, as exemplified by the excretion of mutagens in the urine. Mutagens were extracted from urine with blue rayon, and mutagenic activity was determined in the Ames test, in the presence of an activation system derived from Aroclor 1254-induced rats, and employing the Salmonella typhimurium O-acetylase over-expressing YG1024 bacterial strain. Volunteers consumed three well-cooked beefburgers, whereas a concurrently cooked fourth burger was analyzed for mutagenic activity. Following intake of the burgers, an increase in urinary mutagenic activity was observed, and mutagenic activity was completely excreted within 24 hours. A good correlation was obtained between the intake and excretion of mutagenic activity. The volunteers consumed the same burger meal on two different occasions, once following intake of 10 cups of strong black tea, and the second following intake of a corresponding volume of water. Urine was collected by each volunteer for 24 hours after the meal, and compliance was ascertained utilizing the excretion of p-aminobenzoic acid. The mutagenic ratio, defined as the ratio of urinary mutagenicity over the intake of mutagenicity, was not altered by the black tea, being 0.142 and 0.135 during the water and tea intake, respectively. It is inferred that short-term intake of high levels of black tea, as part of an otherwise normal diet, does not modulate the human metabolism of heterocyclic amines.

Short-term black tea intake modulates the excretion of urinary mutagens in rats treated with 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ): role of CYP1A2 upregulation.

Rats were exposed to black tea (2.5% w/v) as their sole drinking liquid for either 1 day or 1 month, while controls were maintained on water. After this treatment period, all animals received a single oral dose IQ (2-amino-3-methylimidazo-[4,5-f]quinoline), and urine was collected for 48 h. Mutagenic activity of the urine was determined in the Ames test in the presence and absence of an activation system. The excretion of direct-acting mutagens was markedly reduced following tea intake, and was more pronounced after the 1-day treatment. Similarly, both tea treatments suppressed the excretion of indirect-acting mutagens. Furthermore, both tea treatments induced hepatic CYP1A2 activity and expression, but cytosolic glutathione S-transferase activity was only modestly induced in the group of animals receiving tea for 1 day, and only when DCNB (1,2-dichloro-4-nitrobenzene) was used as substrate; glucuronosyl activity was elevated modestly only in the animals receiving the tea for a month. It is concluded that even short-term exposure to black tea is capable of influencing the metabolic fate of IQ, and this is most likely related to the upregulation of CYP1A2.

Ascorbic acid reduces the frequency of iron induced micronuclei in bone marrow cells of mice.

Iron is a potent oxidant that can lead to the formation of genotoxic lipid peroxides. Ascorbic acid, which enhances dietary iron absorption, has been suggested to enhance the oxidant effects of iron and to directly lead to the formation of lipid peroxides. The combined effects of dietary iron and ascorbic acid on genotoxicity were investigated by measuring the frequency of micronuclei in the bone marrow cells of C3H/He mice. In addition, liver iron concentration was measured in all treated groups. Three weeks old mice were fed diets for 3 weeks containing iron at 100 or 300 mg/kg diet in the form of FeSO(4) that were supplemented either with or without ascorbic acid (15 g/kg diet). The results of the bone marrow micronucleus test revealed that the high iron diet resulted in an increased frequency of micronucleated polychromatic erythrocytes (MnPCEs) as compared to low iron. Ascorbic acid supplementation in the low iron diet did not show any effect on incidence of MnPCEs and protected against the increased frequency of MnPCEs induced by the high iron diet. However, liver iron concentration was significantly increased only in the high iron treated and ascorbic acid supplemented group as compared to all other groups. These results demonstrate that ascorbic acid protects against the clastogenic effects of iron.

Evaluation of genotoxic damage of cadmium chloride in peripheral blood of suckling Wistar rats.

The aim of this study was to evaluate possible genotoxic damage of cadmium chloride exposure in suckling rats by means of the comet assay and the in vivo micronucleus test of rat blood lymphocytes, because no information is available on the genotoxic effect of cadmium in rats at this early age. Pups were receiving cadmium (as CdCl(2).H(2)O) orally in fractions of 0.5 mg for 9 days, totalling 4.5 mg Cd kg(-1) body wt, or were given a single subcutaneous injection of 0.5 mg Cd kg(-1) body wt. Some pups in both exposed groups were receiving calcium supplement (CaHPO(4).2H(2)O) in feed to reduce the body load of cadmium. Control pups did not receive either cadmium or calcium supplement. Cadmium in the carcass and organs was measured by atomic absorption spectrometry. The results showed that the cadmium body burden was significantly lower when the animals were receiving calcium supplements along with oral cadmium. The results of the micronucleus and comet assays showed significant differences between the control and exposed groups, regardless of the route of cadmium administration. The only statistically significant difference between the two exposed groups (oral cadmium and oral cadmium + calcium supplements) was in the number of micronuclei. The results of the comet assay showed that tail length differed statistically only between the control and all exposed groups, regardless of the route of cadmium administration. It can be concluded that the applied cadmium doses caused detectable genome damage but it was lower in calcium-treated pups receiving cadmium orally.

Potential health impacts of excessive flavonoid intake.

Plant flavonoids are common dietary components that have many potent biological properties. Early studies of these compounds investigated their mutagenic and genotoxic activity in a number of in vitro assays. Recently, a renewed interest in flavonoids has been fueled by the antioxidant and estrogenic effects ascribed to them. This has led to their proposed use as anticarcinogens and cardioprotective agents, prompting a dramatic increase in their consumption as dietary supplements. Unfortunately, the potentially toxic effects of excessive flavonoid intake are largely ignored. At higher doses, flavonoids may act as mutagens, pro-oxidants that generate free radicals, and as inhibitors of key enzymes involved in hormone metabolism. Thus, in high doses, the adverse effects of flavonoids may outweigh their beneficial ones, and caution should be exercised in ingesting them at levels above that which would be obtained from a typical vegetarian diet. The unborn fetus may be especially at risk, since flavonoids readily cross the placenta. More research on the toxicological properties of flavonoids is warranted given their increasing levels of consumption.

Permeability, cytotoxicity, and genotoxicity of Cr(III) complexes and some Cr(V) analogues in V79 Chinese hamster lung cells.

The permeabilities and genotoxicities of the Cr(III) complexes [Cr(en)(3)](3+), mer-[Cr(glygly)(2)](-), cis-[Cr(phen)(2)(OH(2))(2)](3+), and trans-[Cr(salen)(OH(2))(2)](+) and the Cr(V) analogues of cis-[Cr(phen)(2)(OH(2))(2)](3+) and trans-[Cr(salen)(OH(2))(2)](+) [en being 1,2-ethanediamine, glygly being glycylglycine, phen being 1,10-phenanthroline, and salen being N,N'-ethylenebis(salicylideneiminato)] have been studied in V79 Chinese hamster lung cells. Following exposure of approximately 10(6) cells to 0.4 mM Cr(III) for 4 h, the Cr uptake by single cells was less than 10(-)(14) g/cell (as determined by GFAAS analysis and as confirmed by PIXE analysis where the Cr concentration was below the limit of detection). Importantly, the Cr(V) analogue of cis-[Cr(phen)(2)(OH(2))(2)] was significantly more permeable than the Cr(III) complex. The cytotoxicity of the Cr(III) complexes increased in the following order: mer-[Cr(glygly)(2)](-) < [Cr(en)(3)](3+) approximately cis-[Cr(phen)(2)(OH(2))(2)](3+) < trans-[Cr(salen)(OH(2))(2)](+). No genotoxic effects were observed following exposure to mer-[Cr(glygly)(2)](-) or [Cr(en)(3)](3+) at concentrations up to 6 mM. The Cr(III) imine complexes trans-[Cr(salen)(OH(2))(2)](+) and cis-[Cr(phen)(2)(OH(2))(2)](3+), which could be oxidized to Cr(V) complexes, induced MN in vitro at rates of 13.6 and 3.3 MN/1000 BN cells/micromol of Cr, respectively. The comparative permeabilities and genotoxicities of trans-[Cr(salen)(OH(2))(2)](+) and [CrO(salen)](+) were similar due to the instability of the Cr(V) complex at physiological pH values (7.4). There was a substantial increase in the permeability of [Cr(O)(2)(phen)(2)](+), compared to that of the Cr(III) analogue, which was accompanied by a highly genotoxic response. Consequently, any Cr(III) complex that is absorbed by cells and can be oxidized to Cr(V) must be considered as a potential carcinogen. This has potential implications for the increased use of Cr(III) complexes as dietary supplements and highlights the need to consider the genotoxicities of a variety of Cr(III) complexes when determining the carcinogenic potential of Cr(III) particularly when "high" deliberately administered doses are concerned.

Dietary ortho phenols that induce glutathione S-transferase and increase the resistance of cells to hydrogen peroxide are potential cancer chemopreventives that act by two mechanisms: the alleviation of oxidative stress and the detoxification of mutagenic xenobiotics.

Oxidative stress is implicated in the etiology of cancer, hence compounds that alleviate oxidative stress by inducing enzymes that defend against free radical damage might be useful as cancer chemopreventives. Glutathione S-transferase (GST) has been suggested to be a candidate for a critical enzyme in protecting cells against free radical damage, in part, because its level of induction correlates with protection of the cell line IMR-32 against hydrogen peroxide-induced oxidative stress. The present study identified dietary ortho phenols that both induce GST and protect the cell line IMR-32 against hydrogen peroxide-caused oxidative stress. The ortho phenol (o-phenol) inducers were better protectors against oxidative stress than a number of GST inducers that did not bear phenolic groups, possibly because the phenol residues of the ortho phenols allowed their action as antioxidants as well as inducers of GST. GST has previously been thought to protect cells against cancer by detoxifying mutagenic xenobiotics. The present results suggest that ortho phenol inducers of GST might be useful as cancer chemopreventives that act by two independent mechanisms, the alleviation of oxidative stress and the detoxification of mutagenic xenobiotics.

Genotoxicity of instant coffee: possible involvement of phenolic compounds.

Instant coffee exhibits direct genotoxic activity in the tester strains TA 98, 100, 102, 104 and YG 1024. In the Ames tester strain TA 100, the presence of S9 mix, S100 mix, S9 mix without cofactors led to a significant decrease of the genotoxicity observed. The decrease observed in the presence of S9 mix seems to be highly correlated with the catalase content of S9 mix. The genotoxicity of instant coffee detected in strain TA 100 was dependent on the pH, with higher genotoxic effects at pH values above neutrality. Also, dependent on the pH was the ability of some phenolic molecules present in coffee promoting the degradation of deoxyribose in the presence of Fe3+/EDTA. These results suggest that apart from other molecules present in instant coffee responsible for their genotoxicity in several short term assays, phenolic molecules could also be implicated in the genotoxicity of coffee, via reactive oxygen species arising from its auto-oxidation.

Musk xylene: analysis, occurrence, kinetics, and toxicology.

1,3-Dimethyl-2,4,6-trinitro-5-tert.-butylbenzene (musk xylene, MX), a synthetic musk, is often used in fragrances and soaps to substitute the natural musk. MX belongs to the common group of nitromusk compounds. The main environmental intake of MX occurs after sewage introduction. The consumption of fish and drinking water as well as the use of body care and perfumed household products could lead to an ingestion of this substance in humans. Although the acute oral and dermal toxicity of MX is low, some hint for the carcinogenic potential of MX was found in one animal experiment. These findings and the high potential of MX as environmental contaminant, it is stable against biological and chemical degradation and it is highly lipophil, raised considerable attention in the field of environmental medicine. Biological monitoring and the toxicology of MX, which previously has been described to occur in human milk, human fat tissue, as well as human blood samples, are of central interest. The aim of this article is to summarize the data on the analysis, occurrence, kinetics, and toxicology of MX. As there is a lack of knowledge on human toxicity and human carcinogenicity of MX, a final evaluation of the toxicological data with regard to public health is still impossible. Nevertheless, in view of the published data about MX, there is no evidence for any substantial human risk at the moment.

Inhibition of PhIP mutagenicity by caffeine, lycopene, daidzein, and genistein.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-beta]pyridine NPhIP) is a major dietary component in individuals eating cooked meats or fish. This heterocyclic amine requires biochemical activation, mainly through cytochrome P4501A2, and can be detoxified chiefly by 4'hydroxylation through other cytochromes, and be in turn converted through phase 2 enzymes to readily excreted conjugates. The active form of PhIP is mutagenic in Salmonella typhimurium TA98 and is a useful substrate to study the possible chemoprotective action of phytochemicals. We found that black and green tea depressed the mutagenicity of PhIP in dose-related fashion, and decaffeinated tea was less powerful an inhibitor. This led to the study of caffeine, that displayed effective dose-related inhibition of the mutagenicity of PhIP. Other antioxidants such as lycopene, the active antioxidant from tomatoes, and daidzein and genistein from soy products, also had a dose-related inhibition of the mutagenicity of PhIP. We conclude that PhIP is a good substrate found in several human foods to determine the protective effect of phytochemicals from vegetables, and beverages.

Iron-induced oxidative DNA damage and its repair in primary rat hepatocyte culture.

Iron-overload diseases frequently develop hepatocellular carcinoma. The genotoxic mechanism whereby iron is involved in hepatocarcinogenesis might involve an oxidative process via the intermediate production of reactive oxygen species. This was presently investigated by examining kinetics of formation and repair of DNA base lesions in primary rat hepatocyte cultures supplemented with the iron chelate, ferric nitrilotriacetate Fe-NTA (10 and 100 microM). Seven DNA base oxidation products have been identified in DNA extracts by gas chromatography-mass spectrometry, which showed a predominance of oxidized-purines (8-oxo-guanine, xanthine, fapy-adenine, 2-oxo-adenine) above oxidized pyrimidines (5-OHMe-uracil, 5-OH-uracil, 5-OH-cytosine) in control cultures. All these DNA oxidation products revealed a significant dose-dependent increase at 4 to 48 h after Fe-NTA supplementation, among which fapy-adenine showed the highest increase and 5-OH-cytosine was the least prominent. Involvement of iron in this oxidative process was established by a correlation between extent in DNA oxidation and intracellular level of toxic low molecular weight iron. DNA excision-repair activity was estimated by release of DNA oxidation products in culture medium. All the seven DNA oxidation products were detected in the medium of control cultures and showed basal repair activity. This DNA repair activity was increased in a time- and dose-dependent fashion with Fe-NTA. Oxidized-pyrimidines, among which was 5-OHMe-Uracil, were preferentially repaired, which explains the low levels detected in oxidized DNA. Since oxidized bases substantially differed from one another in terms of excision rates from cellular DNA, specific excision-repair enzymes might be involved. Our findings, however, demonstrate that even though DNA repair pathways were activated in iron-loaded hepatocyte cultures, these processes were not stimulated enough to prevent an accumulation of highly mutagenic DNA oxidative products in genomic DNA. The resulting genotoxic effect of Fe-NTA might be relevant in understanding the hepatocarcinogenic evolution of iron-overload diseases.

Decreased levels of 2-amino-3-methylimidazo[4,5-f]quinoline-DNA adducts in rats treated with beta-carotene, alpha-tocopherol and freeze-dried aloe.

To assess mechanisms of chemoprevention of hepatocarcinogenesis by trans-beta-carotene (beta-C), DL-alpha-tocopherol (alpha-T), and freeze-dried whole leaves of Kidachi aloe (Aloe), formation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-DNA adducts was measured by 32P-post-labeling analysis, and CYP1A1 and CYP1A2 protein levels were analyzed by ELISA. Group 1 rats were fed diet containing 0.02% beta-C, 1.5% alpha-T or 30% Aloe over an 8-day period, while group 2 was given basal diet alone. On day 7, all animals were subjected to two-thirds partial hepatectomy (PH). Twelve hours after PH, they received a single dose of the carcinogenic food pyrolysate IQ (100 mg/kg) intragastrically, to initiate hepatocarcinogenesis. Rats were killed 6, 12, 24 and 48 h after IQ administration. The levels of adducts, expressed as relative adduct labeling values in rats treated with beta-C, alpha-T and Aloe, were decreased as compared with the control group at hour 24 (36 h after PH), with a significant difference in the case of the beta-C group (46.4% of the control value). Similarly, all showed a tendency for decrease at hour 48. Furthermore, the levels of CYP1A2, known to be responsible for activation of IQ, showed a significant reduction at hour 24. It is concluded that beta-C, and possibly also alpha-T and Aloe, have the potential to reduce IQ-DNA adduct formation, presumably as a result of decreased formation of active metabolites. The results may explain, at least in part, the previously observed inhibitory effects of these compounds on induction of preneoplastic hepatocellular lesions.