Aflatoxin B1: A review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification methods.

Aflatoxins are a class of carcinogenic mycotoxins produced by Aspergillus fungi and are known to contaminate a large portion of the world's food supply. Aflatoxin B1 (AFB1) is the most potent of these compounds and has been well-characterized to lead to the development of hepatocellular carcinoma (HCC) in humans and animals. This review focuses on the metabolism of AFB1, including epoxidation and DNA adduction, as it concerns the initiation of cancer and the underlying mechanisms. The link between AFB1 consumption and HCC is also discussed including synergistic interactions with the hepatitis B virus. Toxic effects of AFB1, including growth suppression, malnutrition, and immunomodulation, are also covered. This review also describes recent reports of AFB1 occurrence in global food supplies and exposures in occupational settings. Furthermore, a summary of recent detoxification methods is included to indicate the present state of the field in developing aflatoxin control methods. This information shows that AFB1 occurs frequently in food supplies at high concentrations, particularly in maize. Regarding detoxification methods, chemical control methods were the fastest methods that still retained high detoxification efficacy. The information presented here highlights the need to implement new and/or existing detoxification methods to reduce the global burden of AFB1 toxicity.

Formation and inhibition of N-nitrosodiethanolamine in cosmetics under pH, temperature, and fluorescent, ultraviolet, and visual light.

N-nitrosodiethanolamine (NDELA), a type of nitrosamine, is a possible human carcinogen that may form in cosmetic products. The aim of this study was to examine the formation and inhibition of NDELA through chemical reactions of secondary amines including mono-ethanolamine, di-ethanolamine (DEA), and tri-ethanolamine (TEA), and sodium nitrite (SN) under varying conditions such as pH, temperature, and fluorescent, ultraviolet (UV), and visual light (VIS) using liquid chromatography-mass spectroscopy. In a mixture of TEA and SN under acidic conditions pH 2, residual NDELA concentrations rose significantly under various storage conditions in the following order: 50°C > 40°C > UV (2 W/m2) > VIS (4000 lux) > fluorescent light > 25°C > 10°C. In a mixture of DEA and SN under the same acidic pH 2 conditions, NDELA formation was significantly elevated in the following order: UV (2 W/m2) > VIS (4000 lux) > 50°C > 40°C > fluorescent light > 25°C > 10°C. Inhibition of NDELA formation by d-mannitol, vitamin C (Vit C), or vitamin E (Vit E) was determined under varying conditions of pH, temperature, and fluorescent, UV, and VIS. At high concentrations of 100 or 1000 µg/ml, Vit E significantly decreased residual NDELA compared with control levels under acidic pH 2, but not under basic pH 6. Among various antioxidants, Vit E reacted more effectively with many nitrosating agents such as nitrate and nitrite found in cosmetic products. Therefore, to reduce NDELA, it is recommended that cosmetics be stored under cool/amber conditions and that Vit E or Vit C inhibitors of nitrosation be optimally added to cosmetic formulations at concentrations between 100 and 1000 µg/ml.

Development of a 4-NQO toxic equivalency factor (TEF) approach to enable a preliminary risk assessment of unknown genotoxic compounds detected by the Ames II test in UV/H2O2 water treatment samples.

An approach to enable a preliminary risk assessment of unknown genotoxic compounds formed by MP UV/H2O2 treatment of nitrate rich water, is described. Since the identity and concentration of specific genotoxic compounds is not established yet, a compound specific risk assessment cannot be performed. This limitation is circumvented by introducing a toxic equivalency factor, converting the concentration of unknown genotoxic compounds expressed by an Ames II test response into equivalent concentrations of 4-nitroquinoline oxide (4-NQO), to enable a preliminary risk assessment. Based on the obtained 4-NQO equivalent concentrations for the tested water samples and 4-NQO carcinogenicity data, an indication of the associated risk of the by MP UV/H2O2 treatment produced nitrated genotoxic compounds is obtained via the margin of exposure (MOE) approach. Based on a carcinogen study by Tang et al. (2004), a body weight of 70 kg and a drinking water consumption of 2 L per day, the 4-NQO equivalent concentration should not exceed 80 ng/L associated with a negligible risk. Application of this approach on samples from MP UV/H2O2 treated water of a full scale drinking water production facility, a 4-NQO equivalent concentration of 107 ng/L was established. These results indicate a safety concern in case this water would be distributed as drinking water without further post treatment.

Protein kinase C-activating tumor promoters modulate the DNA damage response in UVC-irradiated TK6 cells.

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a non-genotoxic tumor promoter that dysregulates the protein kinase C (PKC) pathway and causes variable cellular responses to DNA damage in different experimental models. In the present study, we pretreated human lymphoblastoid TK6 cells (wild-type p53) for 72 h with TPA, and five other PKC-activating tumor promoters, to determine how sustained exposure to these chemicals modulates key DNA damage response (DDR) endpoints induced by UVC-irradiation. Here we show that pre-treatment with PKC-activating tumor promoters augmented the sensitivity of TK6 cells to UVC-irradiation characterized by a synergistic increase in apoptosis compared to that induced by either stress alone. In addition, high residual levels of the DNA damage repair signal γH2AX was observed in tumor promoter treated cells indicating a delayed DDR recovery. NH32 (p53-null, isogenic to TK6) cells were resistant to the synergistic effects on apoptosis implicating p53 as a central mediator of the DDR modulating effects. In addition, analysis of p53 target genes in TPA-pre-treated TK6 cells revealed a significant modulation of UVC-induced gene expression that supported a shift toward a pro-apoptotic phenotype. Therefore, sustained exposure to tumor promoting agents modulates the UVC-induced DDR in TK6 cells, which may represent important synergistic interactions that occur during tumor promotion.

Photoirradiation of dehydropyrrolizidine alkaloids--formation of reactive oxygen species and induction of lipid peroxidation.

Pyrrolizidine alkaloid (PA)-containing plants are widespread in the world and are probably the most common poisonous plants affecting livestock, wildlife, and human. PAs require metabolic activation to generate pyrrolic metabolites (dehydro-PAs) that bind cellular protein and DNA, leading to hepatotoxicity and genotoxicity, including tumorigenicity. In this study we report that UVA photoirradiation of a series of dehydro-PAs, e.g., dehydromonocrotaline, dehydroriddelliine, dehydroretrorsine, dehydrosenecionine, dehydroseneciphylline, dehydrolasiocarpine, dehydroheliotrine, and dehydroretronecine (DHR) at 0-70 J/cm2 in the presence of a lipid, methyl linoleate, resulted in lipid peroxidation in a light dose-responsive manner. When irradiated in the presence of sodium azide, the level of lipid peroxidation decreased; lipid peroxidation was enhanced when methanol was replaced by deuterated methanol. These results suggest that singlet oxygen is a photo-induced product. When irradiated in the presence of superoxide dismutase, the level of lipid peroxidation decreased, indicating that lipid peroxidation is also mediated by superoxide. Electron spin resonance (ESR) spin trapping studies confirmed that both singlet oxygen and superoxide anion radical were formed during photoirradiation. These results indicate that UVA photoirradiation of dehydro-PAs generates reactive oxygen species (ROS) that mediated the initiation of lipid peroxidation. UVA irradiation of the parent PAs and other PA metabolites, including PA N-oxides, under similar experimental conditions did not produce lipid peroxidation. It is known that PAs induce skin cancer and are secondary (hepatogenous) photosensitization agents. Our results suggest that dehydro-PAs are the active metabolites responsible for skin cancer formation and PA-induced secondary photosensitization.

Evaluation of tumour promoting potency of fish borne toxaphene residues, as compared to technical toxaphene and UV-irradiated toxaphene.

In this study the potential impact of food chain-based biotransformation and physico-chemical weathering of toxaphene on its tumour promoting potential was investigated in vitro and in vivo. Human exposure to toxaphene is mainly through consumption of contaminated fish, therefore fish-borne residues of toxaphene (cod liver extract, CLE) were prepared by exposing cod to technical toxaphene (TT) for 63 days. UV-irradiated toxaphene (uvT) was included to represent a physico-chemical weathered toxaphene mixture. In vitro, TT, uvT and CLE all showed a dose- and time-dependent inhibition of gap junctional intercellular communication (GJIC) with a relative potency of CLE>TT=uvT. Tumour promoting potency was further studied in vivo in a medium term two-stage initiation/promotion bioassay in female Sprague-Dawley rats, using an increase in altered hepatic foci positive for glutathione-S-transferase-P (AHF-GST-P) as read out. No increase in AHF-GST-P occurred following exposure to either TT, uvT, or CLE, except for the positive control group (2,3,7,8-TCDD). Based on this study the no observed adverse effect level (NOAEL) for tumour promoting potency is at least 12.5mg/kg/week, or higher for CLE. Considering current human exposure levels in Europe it is doubtful that consumption of fish at current levels of toxaphene contamination give rise to human health risk.

Carbon monoxide signalling reduces photocarcinogenesis in the hairless mouse.

Exposure of the skin of mice to UVA (320-400 nm) radiation has been shown to provide protection against the immunosuppressive effects of UVB (290-320 nm) radiation. The UVA protection was mediated via the UVA induction of the stress protein heme oxygenase-1, and its enzymatic product carbon monoxide (CO). Because UVB-induced immunosuppression is an accompanying and prerequisite feature of the promotion phase of photocarcinogenesis, the potential for immunoprotective CO to act as an anti-skin cancer agent was tested in this study. Groups of female albino Skh:hr-1 hairless mice were irradiated chronically with daily minimally erythemogenic doses of solar simulated UV radiation (SSUV) during a 10 week-period to induce photocarcinogenesis. The effect of repeated topical application of lotions containing a CO-releasing molecule (CORM-2; tricarbonyldichlororuthenium (II) dimer) at 250 or 500 microM, that had previously been shown in short-term experiments to provide photoimmune protection in mice, was measured. Tumor development was monitored for 29 weeks. Topical CORM-2 treatment was observed to reduce the acute and chronic inflammatory erythema reaction compared with control irradiated mice that did not receive CORM-2 lotions, and to reduce the chronic epidermal hyperplasia accompanying tumor outgrowth. The CORM-2 treatments provided a significant moderate inhibition of early tumor appearance dose-dependently, significantly reduced the average tumor multiplicity, increased the regression of established tumors dose-dependently, and inhibited the formation of large locally invasive tumors. The CORM-2 treatments also reduced the expression of immunosuppressive IL-10 in the uninvolved epidermis and dermis of tumor-bearing mice, and enhanced immunopotentiating epidermal IL-12 expression. Therefore CO signalling was revealed to have previously unrecognized anti-carcinogenic functions in the skin, consistent with a protective modulation of the epidermal cytokines. This is a novel observation that also implies that the UVA waveband that produces CO physiologically in exposed skin, might likewise be found to have an anti-photocarcinogenic action.

Aqueous photodegradation and toxicity of the polycyclic aromatic hydrocarbons fluorene, dibenzofuran, and dibenzothiophene.

Decay kinetics resulting from the application of UV and UV/H(2)O(2) to the polycyclic aromatic hydrocarbons (PAHs) fluorene, dibenzofuran and dibenzothiophene was studied. Batch experiments were conducted with both low-pressure monochromatic (253.7nm) and medium pressure polychromatic (200-300nm) UV sources alone or in the presence of up to 25mg/L hydrogen peroxide, in a quasi-collimated beam apparatus. Degradation of all three PAHs, by both UV and UV/H(2)O(2), exhibited pseudo-first-order reaction kinetics and low quantum yields ranging from 1.4x10(-3) to 1.8x10(-2)mol/E using both UV lamps. Toxicity testing using a bioluminesence inhibition bioassay was correlated to the decay in concentration of the PAHs as analyzed analytically using HPLC. Results demonstrated that treatment efficacy of oxidative PAH degradation measured by following the decay of the target compound is best complemented by also evaluating the toxicity of the treated water due to byproduct formation concerns.

Stable loss of global DNA methylation in the radiation-target tissue--a possible mechanism contributing to radiation carcinogenesis?

Radiation-induced lymphomagenesis and leukemogenesis are complex processes involving both genetic and epigenetic changes. Although genetic alterations during radiation-induced lymphoma- and leukemogenesis are fairly well studied, the role of epigenetic changes has been largely overlooked. Rodent models are valuable tools for identifying molecular mechanisms of lymphoma and leukemogenesis. A widely used mouse model of radiation-induced thymic lymphoma is characterized by a lengthy "pre-lymphoma" period. Delineating molecular changes occurring during the pre-lymphoma period is crucial for understanding the mechanisms of radiation-induced leukemia/lymphoma development. In the present study, we investigated the role of radiation-induced DNA methylation changes in the radiation carcinogenesis target organ--thymus, and non-target organ--muscle. This study is the first report on the radiation-induced epigenetic changes in radiation-target murine thymus during the pre-lymphoma period. We have demonstrated that acute and fractionated whole-body irradiation significantly altered DNA methylation pattern in murine thymus leading to a massive loss of global DNA methylation. We have also observed that irradiation led to increased levels of DNA strand breaks 6 h following the initial exposure. The majority of radiation-induced DNA strand breaks were repaired 1 month after exposure. DNA methylation changes, though, were persistent and significant radiation-induced DNA hypomethylation was observed in thymus 1 month after exposure. In sharp contrast to thymus, no significant persistent changes were noted in the non-target muscle tissue. The presence of stable DNA hypomethylation in the radiation-target tissue, even though DNA damage resulting from initial genotoxic radiation insult was repaired, suggests of the importance of epigenetic mechanisms in the development of radiation-related pathologies. The possible role of radiation-induced DNA hypomethylation in radiation-induced genome instability and aberrant gene expression in molecular etiology of thymic lymphomas is discussed.

Evaluation of photo-mutagenicity and photo-cytotoxicity of food coloring agents.

Pigments extracted from natural products are widely used for food coloration in Japan. An investigation concerning the photo-mutagenicity and photo-carcinogenicity of frequently used colorants in Japan was performed. Colorants examined were from Laccifer lacca (lac-color), Coccus cacti (cochineal-color), Carthamus tinctorius (carthamus yellow), Gardenia augusta (gardenia yellow and gardenia blue), Monascus anka and Monascus purpureus (monascus red), the skin of Vitis vinifera and Vitis labrusca (grape-skin color), Tamarindus indica (tamarind brown) and Beta vulgaris (beet red). No significant increase in bacterial mutation was found when Salmonella typhimurium TA98, TA100 and TA102 were simultaneously treated with colorants and subjected to UVA irradiation for 30 min. When colorant solutions were subjected to UVA irradiation for 4 h, irradiated solutions containing lac-color became slightly mutagenic toward S.typhimurium TA98 without metabolic activation. A decrease in cell survival resulted when WTK-1 cells were subjected to UVA irradiation for 60 min in the presence of purpurin at 1 mg/ml. Delayed cytotoxicity was also observed following 24 h incubation in fresh medium of samples that were subjected to UVA irradiation for 60 min in the presence of colorant (carthamus yellow, grape-skin color, gardenia blue, cochineal-color, monascus red or purpurin).

Evidence for beneficial low level radiation effects and radiation hormesis.

Low doses in the mGy range cause a dual effect on cellular DNA. One is a relatively low probability of DNA damage per energy deposition event and increases in proportion to the dose. At background exposures this damage to DNA is orders of magnitude lower than that from endogenous sources, such as reactive oxygen species. The other effect at comparable doses is adaptive protection against DNA damage from many, mainly endogenous, sources, depending on cell type, species and metabolism. Adaptive protection causes DNA damage prevention and repair and immune stimulation. It develops with a delay of hours, may last for days to months, decreases steadily at doses above about 100 mGy to 200 mGy and is not observed any more after acute exposures of more than about 500 mGy. Radiation-induced apoptosis and terminal cell differentiation also occur at higher doses and add to protection by reducing genomic instability and the number of mutated cells in tissues. At low doses reduction of damage from endogenous sources by adaptive protection maybe equal to or outweigh radiogenic damage induction. Thus, the linear-no-threshold (LNT) hypothesis for cancer risk is scientifically unfounded and appears to be invalid in favour of a threshold or hormesis. This is consistent with data both from animal studies and human epidemiological observations on low-dose induced cancer. The LNT hypothesis should be abandoned and be replaced by a hypothesis that is scientifically justified and causes less unreasonable fear and unnecessary expenditure.

Photochemical reaction of 7,12-dimethylbenz[a]anthracene (DMBA) and formation of DNA covalent adducts.

DMBA, 7,12-dimethylbenz[a]anthracene, is a widely studied polycyclic aromatic hydrocarbon that has long been recognized as a probable human carcinogen. It has been found that DMBA is phototoxic in bacteria as well as in animal or human cells and photomutagenic in Salmonella typhimurium strain TA102. This article tempts to explain the photochemistry and photomutagenicity mechanism. Light irradiation converts DMBA into several photoproducts including benz[a]anthracene-7,12-dione, 7-hydroxy-12-keto-7-methylbenz[a]anthracene, 7,12-epidioxy-7,12-dihydro-DMBA, 7-hydroxymethyl-12-methylbenz[a]anthracene and 12-hydroxymethyl-7-methylbenz[a]anthracene. Structures of these photoproducts have been identified by either comparison with authentic samples or by NMR/MS. At least four other photoproducts need to be assigned. Photo-irradiation of DMBA in the presence of calf thymus DNA was similarly conducted and light-induced DMBA-DNA adducts were analyzed by 32P-postlabeling/TLC, which indicates that multiple DNA adducts were formed. This indicates that formation of DNA adducts might be the source of photomutagenicity of DMBA. Metabolites obtained from the metabolism of DMBA by rat liver microsomes were reacted with calf thymus DNA and the resulting DNA adducts were analyzed by 32P-postlabeling/TLC under identical conditions. Comparison of the DNA adduct profiles indicates that the DNA adducts formed from photo-irradiation are different from the DNA adducts formed due to the reaction of DMBA metabolites with DNA. These results suggest that photo-irradiation of DMBA can lead to genotoxicity through activation pathways different from those by microsomal metabolism of DMBA.

Photochemical mutagenicity of phototoxic and photochemically carcinogenic fluoroquinolones in comparison with the photostable moxifloxacin.

Certain fluoroquinolone (FQ) antibiotics that show clinical phototoxicity and experimental photochemical carcinogenicity have been found to interact with ultraviolet-A (UVA) radiation to produce oxidative DNA damage in cultured cells and isolated DNA. To study the biological consequences of oxidative DNA damage in mammalian cells, the photochemical mutagenicity of two photoactive FQs, lomefloxacin and Bay y3118, was studied in V79 cells in comparison with that of the photostable moxifloxacin. Lomefloxacin and Bay y3118 were photochemically mutagenic to V79 cells with UVA irradiation, increasing the mutation frequency by about eightfold (400 microM, 6000 J/m2) and tenfold (50 microM, 1000 J/m2), respectively, whereas no photochemical mutagenicity was observed with moxifloxacin (400 microM, 9000 J/m2). We suggest that the previously reported ability of lomefloxacin and Bay y3118 to photochemically produce oxidative DNA damage, which is known to be mutagenic, may be the basis for the photochemical mutagenicity and the reported photochemical carcinogenicity. The photostable moxifloxacin appears to lack such properties.

Paragenetic suppressors of suppressor genes--a new class of oncodeterminants.

Impairment or loss of suppressor genes is a common event permitting the oncogene/suppressor gene machinery to develop neoplasia. Following prenatal treatment with X-rays and UV-B, we detected a new class of oncodeterminants that could not be specified as genes. This points to paragenetic elements that suppress suppressorgenes and thus provoke melanoma at earlier ages of onset as expected, with increased severity and increased number of incidences in successive generations, in the absence of further treatment. These elements were isolated from a xiphophorine DNA library by endogenously labeled long terminal repeats (LTR) of a xiphophorine retrovirus, and were characterized as retrotransposons by Southern and Northern blotting and reverse transcription/polymerase chain reaction and transient transfection studies, in situ hybridization, and sequencing. They appear in multiple copies in the telomeric chromosome regions, where they can extend. Three open reading frames (ORF) are flanked by LTR that contain genetically active regulatory elements, and are inducible by UV-B. ORF 3 shows nests of CG dinucleotides and CGG trinucleotides, which are reminiscent of CGG nests predisposing subjects to anticipation of certain human diseases involving tumor generation. Genetic anticipation as defined by Nettleship (1909) or Warren (1996) including an increase of neoplasia might represent an acquired genetic load in preceding generations, which might provide a lead to a molecular understanding of the worldwide increase of incidences of human tumor.

Effect of 50 Hz sinusoidal electromagnetic field on the kinetics of 14CO2 exhalation after [14C]-N-nitrosodiethylamine administration in mice.

N-Nitrosodiethylamine (NDEA) has been identified as a typical environmental carcinogen. Its metabolism was studied in mice under the influence of an electromagnetic field (EMF). After intraperitoneal administration of [14C]-NDEA, 0.2 microCi/100 g body weight resulted in 22.8% of the total radioactivity exhaled as 14CO2 within 1 h. Mice were exposed to a 50 Hz, 2 mT (rms) electromagnetic field, 8 h/day for 8 weeks. There was a significant increase in the metabolic turnover of [14C]-NDEA into 14CO2 at the end of both 6 and 8 weeks of field exposure, i.e., 26.9% and 37.4% respectively. The enhanced capacity of mice to metabolize NDEA after the exposure to EMF may result in animals with a smaller amount of the bioactive carcinogen burden, thereby indicating a protective role of 2 mT EMF in a whole animal study.

Effect of microwave pretreatment on heterocyclic aromatic amine mutagens/carcinogens in fried beef patties.

To investigate a method to reduce the amount of mutagenic/carcinogenic heterocyclic aromatic amines formed during frying of ground beef, the mutagenic activity in Salmonella strain TA98 was assessed and the amount of known heterocyclic amines was determined by solid-phase extraction and HPLC. The beef patties received microwave treatment for various times before frying. Microwave pretreatment for 0, 1, 1.5, 2 or 3 min before frying at either 200 degrees C or 250 degrees C for 6 min per side reduced heterocyclic aromatic amine precursors (creatine, creatinine, amino acids, glucose), water, and fat up to 30%, in the patties and resulted in a decrease in mutagenic activity up to 95%. The sum of the four heterocyclic aromatic amines shown to be present--2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4,8-trimethylimidazo-[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)--decreased three- to nine-fold compared with control, non-microwaved beef patties fried under identical conditions.

32P-postlabelling studies on the DNA adducts of the food mutagens/carcinogens IQ and PhIP--adduct formation in a chemical system, and by rat and human metabolism.

The DNA adducts of the food mutagens/carcinogens IQ and PhIP were studied by means of 32P-postlabelling techniques. Adducts were generated in vitro and in vivo by three techniques: by photolysis of azido-IQ and azido-PhIP in the presence of dGp, calf-thymus DNA or Salmonella; by administration of IQ and PhIP to rats; and by incubation of cultured COS-1 cells with IQ. These cells expressed the human cytochromes P450 1A1 or P450 1A2 and/or the human N-acetyltransferases NAT1 or NAT2. The data demonstrate that, in the photolytic, rat and human systems, a common IQ metabolite and a common PhIP metabolite are formed together with common sets of IQ adducts and PhIP adducts. The data obtained in the human system show that N-hydroxy-IQ, formed by cytochrome P450, binds poorly to DNA, whereas more efficient binding occurs in the presence of NAT1 and most efficient binding in presence of NAT2. This indicates an O-acetyltransferase activity of human NAT1 and NAT2 and formation of N-acetoxy-IQ as an intermediate and immediate precursor of the ultimate arylnitrenium ion. The effect of the polymorphic NAT2 suggests a critical role for the human acetylation polymorphism in the DNA-binding of IQ in humans and in its genotoxic implications.