Aflatoxin B1: metabolism, toxicology, and its involvement in oxidative stress and cancer development

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

Deciphering Hepatocellular Carcinoma: From Bench to Bedside and Back

Butylated hydroxyanisole (BHA) and beta-naphthoflavone (BNF), both chemicals with anti-carcinogenic properties in some experimental animals, were compared for effects on aflatoxin B1 (AFB1) metabolism, hepatic DNA adduct formation and carcinogenesis in the rainbow trout. Dietary BHA had no effect on the hepatic tumor incidence when fed at 0.03 or 0.3% 4 weeks prior to and during a 4 week dietary exposure of 10 p.p.b. AFB1. BNF, when fed at 0.005 or 0.05% under similar conditions, significantly reduced tumor response, which confirms previous results in trout (Nixon et al., Carcinogenesis, 5, 615-619, 1984). BHA fed at either 0.03 or 0.3% for 8 weeks had no post-initiation effect on the 52 week hepatic tumor incidence of trout exposed to a 0.5 p.p.m. AFB1 solution as embryos. A similar post-initiation exposure to 0.05% BNF significantly enhanced AFB1 tumor response. The influence of dietary BHA and BNF on AFB1 metabolism and DNA adduct formation and persistence in trout were examined. A 3 week pre-treatment with 0.3% dietary BHA had no effect on in vivo hepatic nuclear AFB1-DNA adduct formation at 0.5, 1, 2 and 7 days after AFB1 i.p. injection. By contrast 0.05% dietary BNF reduced hepatic AFB1-DNA adducts to 33-60% of control levels at 0.5, 1, 2 and 4 days after AFB1 exposure. This was accompanied by significantly lower blood and liver levels of AFB1 during the first 24 h after i.p. injection. Livers of BNF trout also contained 4-fold more of the less carcinogenic metabolite, aflatoxin M1, and 50% less aflatoxicol (AFL), a metabolite with similar carcinogenicity as AFB1. Bile AFL-glucuronide levels were significantly decreased in BNF-fed trout, but total bile glucuronides were significantly increased due to a 15-fold increase in aflatoxicol-M1 glucuronide. Freshly isolated hepatocytes from BHA-fed fish, when incubated with AFB1 for 1 h, showed no difference in levels of AFB1-DNA adducts or ratios of AFB1 metabolites when compared to hepatocytes isolated from fish fed a control diet only. By contrast, dietary BNF has been previously shown to greatly enhance AFM1 production, reduce AFL production, and significantly reduce AFB1-DNA adduct formation in isolated trout hepatocytes (Bailey et al., Natl. Cancer Inst. Monograph, 65, 379-385, 1984). These results indicate that dietary BHA up to 0.3% does not alter AFB1 metabolism or DNA adduction in trout, nor does it inhibit or promote AFB1 hepatocarcinogenesis in this species.(ABSTRACT TRUNCATED AT 400 WORDS)

Polychlorinated biphenyls (PCBs) have previously been shown to be inhibitors of carcinogenesis in trout. The mechanism of this inhibition was investigated by studying the effects of PCBs on aflatoxin B1 (AFB1) distribution, metabolism and DNA adduct formation, both in vivo and in vitro. A 24 h distribution study of injected tritiated AFB1 showed more radioactivity in blood, liver and bile in fish fed PCBs, but less in residual carcass. The metabolites of AFB1 found in vivo in blood plasma and liver homogenates were shifted by PCB pretreatment towards greater production of the polar metabolite aflatoxin M1 (AFM1) and glucuronide conjugates. The major metabolite in bile of PCB fish was the glucuronide of aflatoxicol M1 (AFL-M1), which was enhanced 15-fold over controls. Levels of aflatoxicol (AFL) glucuronide, the major conjugate in controls, were unaltered by PCBs. The pattern of AFB1 metabolism in isolated hepatocytes from PCB-prefed fish was consistent with in vivo metabolism. AFB1--DNA adduct formation in a 1 h assay was similar in hepatocytes from PCB-fed and control fish. However, the total rate of AFB1 metabolism was significantly elevated in hepatocytes from PCB-fed fish such that the degree of AFB1--DNA adduct formed per unit AFB1 metabolized was 42% lower than control. Similarly, adduct formation in vivo during the first 24 h post-AFB1 injection in PCB fish was not significantly different from controls. However, over a longer 21 day period, adduct levels in PCB fish were only 48-69% of controls (P less than 0.005, analysis of variance), once peak adduct formation was reached. Thus, initial rates of adduct formation may be misleading in the absence of further information on rates of carcinogen metabolism in vitro and/or pharmacokinetics of peak adduct formation in vivo. These results indicate that PCB inhibition of AFB1 carcinogenesis in trout involves dramatic initial changes in carcinogen distribution, metabolism and elimination which, over time, results in a net reduction of DNA adduct formation.

The American Association for Cancer Research has been the citadel for communicating research on chemical carcinogens for over a century. It therefore seems appropriate that a review of chemical carcinogenesis inaugurates a series of articles highlighting advances in understanding, treating, and preventing cancer.At the dawn of the 20th century, we had recognized that chemicals cause cancer, but we had not yet identified individual cancer-causing molecules, nor did we know their cellular targets. We clearly understood that carcinogenesis, at the cellular level, was predominantly an irreversible process. What we lacked was knowledge of the mechanisms by which chemicals cause cancer and the molecular changes that characterize tumor progression.We now are early in a century in which cancer is being investigated at the molecular level, and we have developed technologies that afford unprecedented power to delineate and manipulate altered pathways in cancer cells. Can we harness new insights and technologies to prevent or obliterate human cancers or delay their progression? Can we identify individuals who have a particularly high susceptibility to specific environmental carcinogens?The history of chemical carcinogenesis is punctuated by key epidemiologic observations and animal experiments that identified cancer-causing chemicals and that led to increasingly insightful experiments to establish molecular mechanisms and to reduction of human exposure. In 1914, Boveri (1) made key observations of chromosomal changes, including aneuploidy. His analysis of mitosis in frog cells and his extrapolation to human cancer is an early example of a basic research finding generating an important hypothesis (the somatic mutation hypothesis). The first experimental induction of cancer in rabbits exposed to coal tar was performed in Japan by Yamagiwa and Ichikawa (2) and was a confirmation of Pott's epidemiologic observation of scrotal cancer in chimney sweeps in the previous century (Fig. 1; ref. 3). Because coal tar is a complex mixture of chemicals, a search for specific chemical carcinogens was undertaken. British chemists, including Kennaway (4), took on this challenge and identified polycyclic aromatic hydrocarbons, for example, benzopryene, which was shown to be carcinogenic in mouse skin by Cook, Hewett, and Hieger in 1933 (5). The fact that benzopyrene and many other carcinogens were polyaromatic hydrocarbons lead the Millers (6) to postulate and verify that many chemical carcinogens required activation to electrophiles to form covalent adducts with cellular macromolecules. This in turn prompted Conney and the Millers (7) to identify microsomal enzymes (P450s) that activated many drugs and chemical carcinogens.The discovery of DNA as the genetic material by Avery, MacLeod, and McCarthy (8) and the description of the structure of DNA by Watson and Crick (9) indicated that DNA was the cellular target for activated chemical carcinogens and that mutations were key to understanding mechanisms of cancer. This led to defining the structure of the principal adducts in DNA by benzo(a)pyrene (10) and aflatoxin B1 (11). The concepts developed in investigating mechanisms of chemical carcinogenesis also led to discoveries that are relevant to other human conditions in addition to cancer, including atherosclerosis, cirrhosis, and aging.Global epidemiologic studies have indentified environmental and occupational chemicals as potential carcinogens. The most definitive epidemiologic studies have been those in which a small group is exposed to an inordinately large amount of a specific chemical, such as aniline dyes.Figure 1 illustrates exposure of individuals to residues from fossil fuel in chimneys, to tobacco smoke, and to fungi containing aflatoxin, and the identification of the responsible carcinogen(s). Active smoking and exposure to second-hand smoke are among the major causes of cancer mortality worldwide. Even after causative chemicals are identified, however, measurement of accumulated exposure of individuals in different environments remains an important challenge.The fact that genetic changes in individual cancer cells are essentially irreversible and that malignant changes are transmitted from one generation of cells to another strongly points to DNA as the critical cellular target modified by tobacco smoke and environmental chemicals. DNA damage by chemicals occurs randomly; the phenotypes of associated carcinogenic changes are determined by selection.Cancers caused by environmental agents frequently occur in tissues with the greatest surface exposure to the agents: lung, gastrointestinal tract, and skin. Recently, the study of chemical carcinogenesis has merged with studies on the molecular changes in cancer cells, thus generating biological markers to assess altered metabolic pathways and providing new targets for therapy. Although these are exciting areas, they may be peripheral to attacking the primary causes of the most common human cancers. As we catalog more and more mutations in cancer cells and more and more changes in transcription regulation, it becomes increasingly apparent that we need to understand what generates these changes. The fact that chemicals cause random changes in our genome immediately implies that our efforts need to be directed to quantifying these changes, reducing exposure, and developing approaches to chemoprevention.Chemical carcinogens cause genetic and epigenetic alterations in susceptible cells imparting a selective growth advantage; these cells can undergo clonal expansion, become genomically unstable, and become transformed into neoplastic cells. This classic view of carcinogenesis has its origin in experimental animal studies conducted in the mid 20th century. The first stage of carcinogenesis, tumor initiation, involves exposure of normal cells to chemical or physical carcinogens. These carcinogens cause genetic damage to DNA and other cellular macromolecules that provide initiated cells with both an altered responsiveness to their microenvironment and a proliferative advantage relative to the surrounding normal cells.Early in the field of chemical carcinogenesis, investigators recognized that perturbation of the normal microenvironment by physical means, such as wounding of mouse skin or partial hepatectomy in rodents (12, 13) or chemical agents, such as exposure of the mouse skin to certain phorbol esters (14), can drive clonal expansion of the initiated cells toward cancer. In the second stage, tumor promotion results in proliferation of the initiated cells to a greater extent than normal cells and enhances the probability of additional genetic damage, including endogenous mutations that accumulate in the expanding population. This classic view of two-stage carcinogenesis (14) has been conceptually important but also an oversimplification of our increasing understanding of the multiplicity of biological processes that are deregulated in cancer. In addition, an active debate continues on the relative contribution of procarcinogenic endogenous mechanisms—for example, free-radical–induced DNA damage (15), DNA depurination (16), DNA polymerase infidelity (17), and deamination of 5-methylycytosine (18)—compared with exposure to exogenous environmental carcinogens (19). The enhancement of carcinogens by epigenetic mechanisms such as halogenated organic chemicals and phytoestrogens (20), as well as the extrapolation of results from animal bioassays for identifying carcinogens to human cancer risk assessment, are also difficult to quantify (21). As discussed below, this debate is not merely an academic one, in that societal and regulatory decisions critical to public health are at issue. The identification of chemical carcinogens in the environment and occupational settings [benzo(a)pyrene and tobacco-specific nitrosamines in cigarette smoke, aflatoxin B1 (AFB1) residues from fossil fuel, vinyl chloride, and benzene] has led to regulations that have reduced the incidence of cancer.A timeline of selected experimental advances in chemical carcinogenesis that have important implications is presented in Fig. 2. First, the selected advances reflect the judgment of the authors and consultants, and remain to be modified by the readers, and, ultimately, by history. Second, the timeline shows the progression of results; an important observation generates new hypotheses that are tested by experiments with increasing mechanistic focus. Third, the timeline is punctuated with three important molecular discoveries (DNA structure, DNA sequence, and the PCR) that refocused experiments in chemical carcinogenesis (9, 22, 23). Fourth, many technological advances have allowed conceptual ideas to be experimentally tested, including the sensitive detection of chemical carcinogens by high-pressure liquid chromatography (24) and mass spectrometry (25), detection of DNA adducts by postlabeling (26) and by specific antibodies (27), transcriptional profiling by arrays (28, 29), and quantitation of mutagenicity of carcinogens using bacterial genetics (19).In the first half of the 20th century, the experimental focus was on identifying chemical carcinogens in complex mixtures, and on determining their metabolism and cellular targets. With the recognition that genes are encoded in DNA (9) and that DNA is transferred from one cellular generation to the next (30), research rapidly focused on the interaction of activated chemical carcinogens with DNA and on mutations that result from DNA alterations as well as the identification of key mutated (31) or deregulated genes including oncogenes and tumor suppressor genes (32). Underlying these studies was the expectation that delineation of mutated genes would identify them as specific targets for chemotherapy. The expectation that targeting individual mutated or rearranged gene products would be efficacious for cancer treatment has thus far been verified in only a limited number of situations, such as the use of imatinib for chronic myelogenous leukemia (33).The experimental landmarks highlighted in Fig. 2 frequently generated new experiments, and this progression has foretold some of our key concepts on the mechanisms of chemical carcinogenesis. An overriding concept has emerged that links DNA damage by reactive chemicals, the production of mutations by unrepaired DNA adducts, and the selection of cells harboring mutated genes that characterize the malignant phenotype. Studies on arylhydroxylamines provided a paradigm for tracing the metabolism of carcinogens to chemically reactive electrophiles that covalently bind to DNA. 2-Acetylaminofluorene (AAF) is metabolically activated by liver microsomal mixed–function oxygenases to N-hydroxy- and then to N-sulf oxy-AAF, a strong electrophile that forms covalent adducts with guanine moieties in DNA (34). AAF is not mutagenic in bacterial assays, whereas N-hydroxy-AAF is highly carcinogenic (34). N-hydroxy-AAF is rendered inactive by the formation of a glucuronide in the liver that is transported to the bladder and excreted (35). Unfortunately, it is subjected to acid hydrolysis in the bladder to yield active N-hydroxy-AAF, which is associated with human bladder cancer. Thus, the activation and detoxification of a chemical carcinogen in specific cells or tissues can be a major factor in determining tissue and host specificity.The testing of certain concepts in chemical carcinogenesis awaited the development of new technologies. For example, the concept of somatic mutations in cancer (1, 36) preceded by 40 years the establishment of DNA as the genetic material (8) and by 63 years the development of DNA sequencing methods (23) that directly showed clonal mutations in human cancer cells. Also, the mutator phenotype hypothesis formulated in 1974 (17) has been only recently experimentally verified (37).Many hypotheses are still under active investigation. These include the potential importance of carcinogen-protein interactions (38), carcinogen-induced reversion to stem cell–like phenotypes (39), inherited changes in gene expression (40, 41), direct action of nongenotoxic chemicals (42), and targeted interactions of carcinogens with specific genes such as TP53 (43–45). Other concepts focus on carcinogenesis mediated by RNA damage (46), RNA-templated DNA repair (47), specific metastasis genes (48, 49), and sequential clonal lineage pathways in cancer (50, 51).Emerging hypothesis such as anticarcinogens (52), overlapping pathways to malignancy (53), coordinated changes in gene expression (54), epigenetic silencing by chemical carcinogens (40, 55, 56), and oncogene addiction (57) are just beginning to be explored. Finally, there are concepts for which quantitation is lacking, yet have stood the test of time based on their inherent significance; these include the importance of anaerobic metabolism by tumors (58, 59) and the initiation of tumorigenesis by the generation of oxygen-reactive species (15).Although establishing DNA as the genetic material provided a structure that faithfully can be duplicated during each cell division, it rapidly became apparent that DNA was also subject to direct modification by X-rays (60), alkylating agents (61), and by an increasing number of environmental chemicals (62, 63). Changes in DNA by many chemical carcinogens are indirect; they first require activation by P-450 aryl hydroxylases into electrophiles to form covalent adducts with DNA and with other cellular macromolecules (64, 65). Many normally generated reactive molecules that are intermediates in metabolism modify many cellular molecules including DNA and therefore are mutagens and carcinogens. However, not all mutagens seem to be carcinogens. What was unanticipated was the magnitude of DNA modification by normal cellular processes in the absence of exposure to environmental mutagens (66, 67).The lability of DNA in an aqueous environment was first quantified by Lindahl and Nyberg, who measured the rates of depurination (16) and deamination (18) in solution under different conditions and extrapolated these results to those predicted to be present in human cells. They calculated that each normal cell could undergo >10,000 DNA damaging events per day. Endogenously generated modifications of DNA include methylation by S-adenosylmethione, modification by lipid peroxidation products, chlorination, glycosylation, oxidation, and nitrosylation (66–71). Reactive oxygen and nitrogen species are particularly relevant because the activated species are generated by host cells, and the process of resynthesis results in the replacement of >50,000 nucleotides per cell per day (68). To maintain our genomes, we have evolved a network of DNA repair pathways to excise altered residues from DNA (Fig. 3). A major consideration is the relative contribution of environmental and endogenous DNA damage to carcinogenesis. DNA damage by environmental agents would have to be extensive and exceed that produced by normal endogenous reactive chemicals to be a major contributor to mutations and cancer. This consideration underlines the difficulty in extrapolating risk of exposure to that which would occur at very low doses of carcinogens.Human cells possess an armamentarium of mechanisms for DNA repair that counter the extensiveness of DNA damage caused both by endogenous and environmental chemicals. These mechanisms include base excision repair (BER) that removes products of alkylation and oxidation (72–74); nucleotide excision repair (NER) that excises oligonucleotide segments containing larger adducts (75); mismatch repair that scans DNA immediately after polymerization for misincorporation by DNA polymerases (76); and oxidative demethylation (77), transcription-coupled repair (TCR) that preferentially repairs lesions that block transcription (78); double-strand break repair and recombination that avoids errors by copying the opposite DNA strand (79); as well as mechanisms for the repair of cross-links between strands (80, 81) that yet need to be established.Most DNA lesions are subject to repair by more than one pathway. As a result, only a minute fraction of DNA lesions escapes correction are present at the time of DNA replication and can direct the incorporation of noncomplementary nucleotides resulting in mutation (Fig. 3). Unrepaired DNA lesions initiate mutagenesis by stalling DNA replication forks or are copied over by error-prone trans-lesion DNA polymerases (82–84). Alternatively, incomplete DNA repair can result in the accumulation of mutations and mutagenic lesions, such as abasic sites (85).Damage to DNA by chemical carcinogens activates checkpoint signaling pathways leading to cell cycle arrest and allows time for DNA repair processes. In the absence of repair, cells can use special DNA polymerases that copy past DNA adducts (86, 87), or undergo apoptosis by signaling the recruitment of immunologic and inflammatory host defense mechanisms. The demonstration that each methylcholanthrene-induced tumor has a unique antigenic signature provided one of the earliest glimpses into the stochastic nature of cellular responses to carcinogens (88). The immunologic and inflammatory responses facilitate not only engulfment and clearance of damaged cells but also the resulting generation of reactive oxygen (89) and nitrogen radicals (90) that further damage cellular DNA.The concept that chronic inflammation can result in cancer is supported by Virchow's (91) histologic observation of inflammatory lymphocytes infiltrating tumors. Inflammation accompanying the "painting" of coal tar was described by Japanese pathologists in the earliest experimental study of chemical carcinogenesis (2). The classic tumor promoter, croton oil, and its most active ingredient, 12-O-tetradecanoylphorbol-13-acetate, are potent inflammatory agents. In addition to studies of "two-stage" skin carcinogenesis, other animal models have shown the synergistic interaction of chemical carcinogens with proinflammatory agents; for example, respiratory infection with influenza virus synergistically increases the lung cancer response in rats to a carcinogenic N-nitrosamine (92).Chronic inflammation can have a strong inherited basis, e.g. hemochromatosis, or can be acquired from infection by viruses, bacteria, or parasites or be associated with metabolic or physical conditions (93). Obesity has been considered to be a chronic inflammatory condition associated with multiple types of human cancer (94); gastric acid reflux causes chronic inflammation and can progress to Barrett's-associated esophageal adenocarcinoma (95); and colitis can progress to colon cancer (96, 97). Recent advances have begun to uncover the underlying mechanisms of the association between chronic inflammation and cancer.The identification of specific genes by allelic replacements and "knockouts" has facilitated the delineation of complex immune response networks that govern cellular responses to chemical carcinogens. The innate immune system is the first line of defense against pathogenic microorganisms and toxins and responds by generating free radicals, inflammatory cytokines, and the activation of the complement cascade (93, 98). In addition to reactive oxygen species, the past two decades have shown the significance of nitrogen-based free radicals, including nitric oxide and its derivatives (90, 93). The concentration and length of exposure can determine the seemingly paradoxical procarcinogenic and anticarcinogenic activities of free As be discussed in another in the chronic activation of the innate immune system is procarcinogenic and immune system is anticarcinogenic there is a to from an individual is exposed to a carcinogen to the detection of a For most there is an in cancer incidence as a of that tumor progression in a series of sequential This process has been most in colon cancer, with the progression from to to and to metastasis of cancers at different from to a sequential of mutations and genome mutations in DNA activation of of on and of This concept of sequential mutations has been by new including the of somatic mutations in and colon cancers and the demonstration that only a small fraction of colon cancers the three most frequently identified mutations this may identify potential not cancers a mutator a more stochastic cancer cell in a tumor of different and yet only a small of cells preferentially during to random mutations that a selective advantage for this concept is the demonstration that the of mutations in human cancers is greater than that in normal tissues in cell and adenocarcinoma of the colon The genetic of cancer cells produced by mutator mutations increases the that a tumor many cells to and is with the of of research in chemical carcinogenesis have provided a for the analysis of adducts and somatic mutations in as of carcinogen exposure. A paradigm for between of carcinogens exposure and a cancer risk is shown in Fig. a is a example of an environmental chemical carcinogen that has been using this a polycyclic aromatic (53), an aromatic and a tobacco-specific N-nitrosamine are other key epidemiologic studies a association between exposure and the incidence of studies of in multiple animal species, chemical and analysis of the identification of DNA adducts, and of mutagenic the for and to as a human carcinogen from these experimental animal and studies were then and to assess exposure and biological in studies conducted in of high exposure and high incidence of such as and The were and to the of the that is a human The between and was further by the association between exposure and a specific mutation in the nucleotide of of the tumor suppressor gene in In from and The a synergistic interaction between exposure or and of virus infection in the risk of was remain to be For example, the molecular of the synergistic interaction between and is still the and oxidative of to the gene incorporation in the genome of their of by advances in molecular are and they increasingly are being to understanding the interaction of chemical carcinogens with cellular and of DNA has facilitated the identification of specific genes mutated in human cancers. including mass to carcinogen with unprecedented and spectrometry is being with mutagenesis to specific alterations in DNA of the human genome and the identification of DNA enzymes the field of molecular in on individual susceptibility to carcinogens. analysis of carcinogen-induced alterations in the expression of both and the are that can molecules of carcinogens in cells, random mutations in individual cells, analysis of the of molecules and and and genetic to delineate complex pathways in cells. Underlying this progress in understanding chemical carcinogenesis is a cascade of advances in molecular that it to quantify DNA damage by chemical agents, and changes in gene the structure of DNA and the cell including carcinogenesis. in detection of DNA damage, including postlabeling of DNA (27), and mass spectrometry (25), have allowed the detection of a altered base in nucleotides using human DNA. This can be to DNA or RNA in a cell in cell including and it to assess changes in RNA and expression during carcinogenesis. these technologies it increasingly to pathways in cancer cells from to to to have made in identifying chemical carcinogens and their mechanisms of We have increasingly focused on DNA as a the fact at the cellular level, cancer is an inherited a cancer, a cancer. The efforts to chemicals as potential or human carcinogens are not but in most are in The need to identify chemical carcinogens in of human exposure and epidemiologic is on mechanistic and knowledge of and among animal species is a For example, the of in the by a not to be relevant to carcinogenesis, is initiated by epidemiologic verified by animal experiments, and by mechanistic and studies The between carcinogen exposure and the induction of cancer continues to be a of and public debate The of a is a in the of public health that to be as mechanistic accumulate in the field of chemical carcinogenesis has a history of that of cancer cancer risk assessment, public health and and occupational causes of cancer. The concepts of interactions and in the molecular of human cancer risk were generated by the of chemical carcinogenesis, cellular and molecular and cancer genetic in DNA repair and enzymes are of an inherited of in cancer susceptibility of the of cancer risk and detection are based on the knowledge of chemical carcinogenesis, including adducts, somatic and mutation carcinogen exposure and DNA with interactions can have synergistic for example, and in carcinogenesis. models of chemical carcinogenesis to a critical in the field of cancer and in our understanding the mechanisms of cancer and the of in in the field of chemical carcinogenesis remain to be stem cells mutated by chemical carcinogens and become of human chemical carcinogens epigenetic changes during These and other many to be formulated by to investigators in chemical carcinogenesis our understanding of carcinogenesis, and, as a result, cancer and potential of were Cancer and and by The Research of the Cancer for Cancer Research of of this were in by the of This therefore be in with to this selection of the major events in this review of the field are the primary of the with the of the The authors for many of which are of importance to the field of chemical carcinogenesis. We on this subject We for Fig. of chimney and for Fig. and of smoking and aflatoxin, and and for their critical

Aflatoxins, a carcinogenic group of mycotoxins, are naturally occurring toxic fungi that cause illness in both animals and humans. Predominantly found in hot and humid areas, aflatoxins are generated by Aspergillus fungi and are found in a large percentage of the world’s food supply. Aflatoxin B1 (AFB1), being the most potent of the over 18 aflatoxins discovered, is most noted for its role in the development of hepatocellular carcinoma (HCC) in humans and animals, unfortunately, many features of the illnesses it causes and the mechanisms that produce them, remain unexplained. This review examined AFB1 metabolism; its epoxidation and DNA adduction, its correlations to cancer initiation and the mechanisms that underpin it, the synergistic interactions with stunted growth associated with AFB1 intake and kwashiorkor, involvement of oxidative stress and reactive oxygen species. Its harmful effects, including growth retardation, starvation, and immunotoxicity, were also discussed, delving into new findings of AFB1 contamination in worldwide food sources. This review indicated that AFB1 is commonly found in high concentrations in food supplies, notably in maize. To lessen the global burden of AFB1 toxicity, data gathered through this review emphasized the necessity to apply novel and existing techniques to prevent these toxins on other diseases.

Aflatoxin B1 Transfer and Metabolism in Human Placenta

Pregnant women have been preforming work-related activities during pregnancy since time immemorial, from the traditional hunter-gatherer or forager society to today’s modern world. But ever since our society has been industrialized, exposure patterns for pregnant women have changed dramatically, and they keep changing. This change is due partly to exposure changes overall in the labor market and partly the changes in the gender balance in different occupations. To some extent, women have been protected from the most hazardous occupations, since these mainly have been held by men (1). But active strategies within Europe to move towards a gender-balanced work force have increased the number of women seeking employment in hazardous work environments. One example is heavy truck and lorry drivers, who are predicted to be 40% female by 2030 (2). Today, the labor participation rate for women is high, with a total of 67% in Europe and 74–80% in Scandinavian countries (3), leading to a workforce that eventually has as many exposed women as men. A gender-balanced work force across occupations brings diversity and thus benefits to the work site (4). But it also introduces a challenge, especially in exposed blue-collar occupations. So far, much emphasis has been given to chemical and particles exposure. This is a very important and crucial area for the risk assessment for pregnant workers (5, 6) but not the only hazard present in occupational settings. Too little attention has been paid to physical factors in the work environment during pregnancy in association with health effects, even though physical factors are more prevalent than chemical and particles exposure in occupational settings. The exception is physical load, about which several original articles and reviews have been published (7–9). But physical exposure is a broad concept and also includes exposure to temperature, whole-body vibration, and noise. Among the physical exposures in occupational settings, physically strenuous work has been the most studied. This area includes work postures, heavy lifting, standing/walking, sedentary work as well as a cardiovascular strain from physical labor. Recent reviews show an overall modest effect for physically strenuous work during pregnancy as well as pregnancy complications and adverse birth outcomes (10). In summary, the evidence so far concludes that pregnant workers should avoid occasional heavy lifting and lifting >10 kg in general (9). Heat in residential settings during pregnancy has been extensively studied (11), and some studies have also focused on occupational settings. An association between heat exposure and heat stress in relation to pregnancy complications as well as birth outcomes has been reported (12). But the evidence is not strong enough to recommend specific temperature levels in occupational settings for pregnant workers. Only a few studies have investigated noise and whole-body vibration and its effect on pregnant workers. In both areas, reviews have shown inconclusive results (13, 14) and, since then, only a handful studies have been published, including only one large scale cohort study divided into five manuscripts (15–19). The evidence so far suggests that working full-time (8 hours) in weighted average of >80dBA occupational noise and >0.5m/s2 whole-body vibration is associated with an increased risk of pregnancy complications and negative birth outcomes. But since these findings come from one cohort, they need to be confirmed in other high quality cohort studies where levels of exposure can be assessed. An important review on the evidence so far regarding physical exposures during pregnancy and preterm birth is on the way; the method and protocols have already been published (20), but a review is only as good as its included studies, and we need more high-quality original studies in this research area. Overall, more prospective cohort studies with objectively assessed exposures are needed to be able to identify a safe level for occupational physical exposures during pregnancy. So far, the current evidence on physical load is predominantly based on self-reported physical load, and future studies should focus on high quality objective exposure assessment to increase the level of evidence within this part of the research field. To be able to progress, future studies also need detailed information on absence from work. In many countries, absence from work due to pregnancy benefit, sick leave and parental leave is common during pregnancy. In Sweden, 7 out of 10 women apply for leave of absence benefits at some point during pregnancy [21). In contrast to chemical exposures, that can bioaccumulate, physical exposures predominately affect the working mother during pregnancy. Hence, to correctly assess occupational exposure to physical factors during pregnancy in relation to health effects in the mother and child, absence from work needs to be addressed properly. Associations can otherwise be missed due to misclassification of exposure. This have been shown in a large-scale cohort study from Sweden, where associations only were found in full time workers with low leave of absence during pregnancy [16-18). Leave of absence data is also needed to assess the potential beneficial effect that leave of absence can have on the pregnant worker. This was shown in Skröder et al 2021, where pregnant women highly exposed to whole-body vibration only had an increased risk of preterm birth if they had few days of leave of absence during pregnancy. With increased levels of leave of absence from work, women highly exposed to whole-body vibration at work had the same risk of delivering preterm as the general working population. In high levels of leave of absence data, women highly exposed to whole-body vibration had a lower risk of preterm birth then the general population with the same level of absence from work, indicating a healthy worker effect [16). Leave of absence during pregnancy can also be seen as an intermediate measure, since early health effects can lead to an increased level of sick leave, parental leave or pregnancy benefit. An association between exposure at the workplace and level of absence from work have been seen in a recent Danish study and in a recent review [22, 23). High quality environmental epidemiological studies have been of use when assessing the effect of occupational exposure during pregnancy to chemical and particle exposure on pregnancy complications and birth outcomes. But for physical exposures, the exposures and levels differ substantially between environmental and occupational settings and some of the mechanisms. This is true for the mechanism between noise and health, where occupational and residential exposure to noise both are associated with a stress mechanism, but were only the residential exposure contribute to sleep disturbance and related health effects [13). An exposome approach is needed to identify and assess all the potential risk factors for pregnant women [24). In the exposome concept, multiple exposures in a life course perspective are assessed, with a focus on vulnerable stages, such as pregnancy. It is important to adjust for other co-exposures in the work environment, such as chemical and particle exposure, psychosocial exposures, shift work and other physical exposures when assessing the relationship between occupational exposure to one physical factor and the outcome. These exposures are partly correlated with each other [15-19). Few previous studies have been able to adjust for other occupational exposures at work when investigating individual occupational exposures. Very few studies examined the interaction of occupational exposure and how they jointly contribute to the risk of health effects in the mother and child, even though most of these exposures correlate in the work place [25). Some of the existing birth cohort studies can be regarded as hidden treasures for occupational data, with detailed information on occupational environment of the parents that many times have not been used [26). We need to be able to produce valid exposure-response curves and thereby ensure a safe work environment for the pregnant worker and her child. But also, to avoid excluding women from the labor market during pregnancy unnecessary. An unnecessary exclusion from the labor market can hinder female workers career advancement to the next level of their career and lead to lower salaries, and in the end lower pensions compared to men. So, a well-balance discussion based on high quality evidence can provide a safe and non-discriminatory work environment for pregnant women. Regarding chemical and particle exposure, an equal low level of exposure for both men and women in reproductive ages can reduce reproductive effects successfully. In physical exposures, there is less need to reduce exposure levels for all, unless these are associated with other health outcomes. It is mainly women of reproductive age who need to be protected, preferably early on since at least whole-body vibration is suspected to be associated with miscarriages. A better system to identify and inform pregnant workers already at the time of their prenatal care registration (usually in gestational week 10) is needed. Overall, there remains a significant knowledge gap regarding the effect of physical occupational exposures during pregnancy and health effects among children. More high-quality cohort studies with objectively assessed exposure that have access to leave-of-absence-during-pregnancy data are needed to increase the level of knowledge in this important

Introduction. By non-invasively cooling the air exhaled by a subject, it is possible to collect a liquid composed primarily of water and a small amount of fluids from the lining of the airways,1 namely the exhaled breath condensate (EBC). This matrix finds use in various applications, including biomarker identification, inflammation assessment, and early diagnosis in at-risk individuals. Recently, some studies have started measuring the concentration of particulate matter in EBC as a tool for assessing the exposure in occupational and environmental settings. However, they often reach conflicting conclusions, partly due to the wide variability of the analytical techniques used to analyze the particulate matter. Objectives. The main contributions of the literature on the use of EBC as a representative matrix for the assessment of particulate matter exposure in occupational settings will be illustrated. Additionally, a pilot study conducted in an office work environment (i.e., a scenario with low exposure to airborne particles) will be presented, using electron microscopy as the quantitative characterization technique for the particulate matter, representing an element of innovation compared to the existing studies. Methods. The most recent publications in which the EBC is used to estimate the occupational exposure to airborne particles have been identified. For the pilot study, the EBC was sampled from several volunteers at the beginning and end of their work shifts, following the existing recommendations to limit contamination and ensure proper sample handling. Information was also obtained regarding the activities carried out during the work shift. Additionally, respirable particles were collected by sampling every subject breathing zone, via personal samplers, for 4 working hours. Airborne respirable particles and those in the EBC were quantified and morphologically/chemically characterized using scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy. Results and Conclusions. The existing publications highlight the need for further steps before the EBC can be properly positioned as a scientific research and occupational exposure monitoring tool. Indeed, the results of some studies are contradictory and lack information at the level of the individually characterizable particles, making them difficult to interpret. Furthermore, in the available literature, the concentration and type of airborne particles are rarely correlated with those present in EBC: starting from the objective of investigating this relationship through a technique such as electron microscopy, which represents the state of the art in terms of resolution and quality of information obtainable about individual particles, the pilot study presented in this work revealed that EBC is a highly complex matrix, subject to interactions between its liquid component and the various airborne particle species to which the subjects are exposed. Indeed, the particles in the EBC are not a simplistic representation of the airborne particle species, instead they must be interpreted in light of the physicochemical characteristics of the inhaled particles, such as their solubility. Precisely for this reason, the amount and type of the particulate matter found in the EBC samples does not seem to be influenced by the smoking status of some of the participants in the pilot study, confirming what was highlighted by previous studies. The “particle-by-particle” analysis approach developed in this study can be further applied to other occupational exposure scenarios to confirm its validity as a method capable of providing information regarding exposure to other particulate matter forms, such as microplastics and wood dust.

CYP1A Induction by β-Naphthoflavone, Aroclor 1254, and 2,3,7,8-Tetrachlorodibenzo-p-dioxin and Its Influence on Aflatoxin B1Metabolism and DNA Adduction in Zebrafish (Danio rerio)

The effect of feeding piglets with the diet containing green tea extracts or coumarin on in vitro metabolism of aflatoxin B1 by their tissues

Rainbow trout (Salmo gairdneri) and coho salmon (oncorhynchus kisutch) were exposed to aflatoxin B1 (AFB1) either by passive embryo uptake or by dietary treatment after hatching and feeding onset. Trout exposed as embryos to an aqueous solution of 0.5 p.p.m. AFB1 for 15 min showed a 62% tumor incidence 12 months later, whereas coho salmon exposed to a similar solution for 30 min showed only a 9% incidence. The difference between salmon and trout response was even greater by dietary AFB1 treatment. Trout exposed for 4 weeks to 20 p.p.b. dietary AFB1 had a 62% tumor response 12 months later, whereas salmon exposed to 40 p.p.b. dietary AFB1 for 4 weeks failed to develop tumors. A 5% tumor incidence was observed in salmon 12 months after 3 weeks exposure to 5000 p.p.b. dietary AFB1, a lethal dose for trout. In addition to a lower tumor incidence when compared to trout, the neoplastic response of salmon to AFB1 is to produce benign hepatic adenomas in contrast to the malignant hepatocellular carcinomas seen in trout. AFB1 metabolism, DNA adduct formation, adduct persistence in vivo and in vitro and cytochrome P-450 isozyme composition were compared in livers of trout and salmon to understand the role of metabolism and initiation in this species difference. AFB1-DNA binding was 7-56 times greater in trout than salmon liver at various times after AFB1 injection, 20 times greater in embryos or in freshly isolated trout hepatocyte preparations after a 1 h incubation with aflatoxin B1, and 18 times greater in trout liver after a three week dietary (80 p.p.b.) exposure. The major AFB1-DNA adduct was 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 in both species. Persistence of AFB1-DNA adducts in vivo in liver was high compared to mammalian systems, implying that active enzymatic removal of bulky DNA adducts is low in both species and probably not a factor in their differential response to aflatoxin. Species differences in other phase I and phase II metabolism pathways and in AFB1 elimination were, overall, much less striking than those previously observed for trout fed inhibitors of aflatoxin carcinogenesis. Rates of bile elimination of AFB1 detoxication products, and total excretion of aflatoxins into water after AFB1 exposure, were not significantly different between trout and salmon. Since detoxication differences were not observed, the species difference in AFB1-DNA binding appears to reflect less efficient cytochrome P-450 metabolism of aflatoxin to the reactive 8,9-epoxide in salmon, compared to trout.(ABSTRACT TRUNCATED AT 400 WORDS)

Although the adverse health consequences of ingestion of food contaminated with aflatoxin B1 (AFB1) are known, relatively few studies are available on the adverse effects of exposure in occupational settings. Taking this into consideration, our study was developed aiming to elucidate the possible effects of occupational exposure to AFB1 in Portuguese swine production facilities using a specific biomarker to assess exposure to AFB1. In total, 28 workers participated in this study, providing blood samples, and a control group (n = 30) was composed of subjects without any type of agricultural activity. Fungal contamination was also studied by conventional methods through air, surfaces, and new and used floor coverage. Twenty-one workers (75%) showed detectable levels of AFB1 with values ranging from <1 ng/ml to 8.94 ng/ml and with a mean value of 1.91 ± 1.68 ng/ml. In the control group, the AFB1 values were all below 1 ng/ml. Twelve different Aspergillus species were identified. Aspergillus versicolor presented the highest airborne spore counts (3210 CFU/m3) and was also detected in higher values in surfaces (>300 CFU/cm2). Data indicate that exposure to AFB1 occurs in swine barns, and this site serves as a contamination source in an occupational setting.

Although there is an abundance of literature concerning the ingestion of food contaminated with aflatoxin B1 (AFB1), only a small number of studies explore mycotoxin exposure in occupational settings. Taking this into consideration, our study was developed with the intention of elucidating whether there is occupational exposure to AFB1 in Portuguese poultry and swine production facilities. A specific biomarker was used to assess exposure to AFB1. A total of 45 workers (34 from poultry farms; 11 from swine production facilities) participated in this study, providing blood samples. Additionally, a control group (n=30) composed of subjects without any type of contact with agricultural activity was considered. All participants signed a consent form and were provided with the study protocol. Eighteen poultry workers (58.6%) and six workers from the swine production facilities (54.5%) showed detectable levels of AFB1. In the control group, the AFB1 values were all below 1 ng/ml. No significant differences in AFB1 levels in serum between workers from poultry and swine farms were found. Poultry workers, however, showed the highest serum levels and a significant statistical difference between this group and the control group was found. Results suggest that exposure to AFB1 by inhalation occurs in both occupational settings representing an additional risk that needs to be recognised, assessed and prevented.

Cytochrome P450 2A13 mediates aflatoxin B1-induced cytotoxicity and apoptosis in human bronchial epithelial cells