Correction to: Mode of action-based risk assessment of genotoxic carcinogens.

The author would like to thank N. Bakhiya, S. Hessel-Pras, B. Sachse, and B. Dusemund for their support in the chapter about pyrrolizidine alkaloids.

Mode of action-based risk assessment of genotoxic carcinogens.

The risk assessment of chemical carcinogens is one major task in toxicology. Even though exposure has been mitigated effectively during the last decades, low levels of carcinogenic substances in food and at the workplace are still present and often not completely avoidable. The distinction between genotoxic and non-genotoxic carcinogens has traditionally been regarded as particularly relevant for risk assessment, with the assumption of the existence of no-effect concentrations (threshold levels) in case of the latter group. In contrast, genotoxic carcinogens, their metabolic precursors and DNA reactive metabolites are considered to represent risk factors at all concentrations since even one or a few DNA lesions may in principle result in mutations and, thus, increase tumour risk. Within the current document, an updated risk evaluation for genotoxic carcinogens is proposed, based on mechanistic knowledge regarding the substance (group) under investigation, and taking into account recent improvements in analytical techniques used to quantify DNA lesions and mutations as well as "omics" approaches. Furthermore, wherever possible and appropriate, special attention is given to the integration of background levels of the same or comparable DNA lesions. Within part A, fundamental considerations highlight the terms hazard and risk with respect to DNA reactivity of genotoxic agents, as compared to non-genotoxic agents. Also, current methodologies used in genetic toxicology as well as in dosimetry of exposure are described. Special focus is given on the elucidation of modes of action (MOA) and on the relation between DNA damage and cancer risk. Part B addresses specific examples of genotoxic carcinogens, including those humans are exposed to exogenously and endogenously, such as formaldehyde, acetaldehyde and the corresponding alcohols as well as some alkylating agents, ethylene oxide, and acrylamide, but also examples resulting from exogenous sources like aflatoxin B1, allylalkoxybenzenes, 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), benzo[a]pyrene and pyrrolizidine alkaloids. Additionally, special attention is given to some carcinogenic metal compounds, which are considered indirect genotoxins, by accelerating mutagenicity via interactions with the cellular response to DNA damage even at low exposure conditions. Part C finally encompasses conclusions and perspectives, suggesting a refined strategy for the assessment of the carcinogenic risk associated with an exposure to genotoxic compounds and addressing research needs.

Human relevance of follicular thyroid tumors in rodents caused by non-genotoxic substances.

Chronic stimulation of the thyroid gland of rodents by TSH leads to thyroid follicular hyperplasia and subsequently to thyroid follicular adenomas and carcinomas. However, the interpretations of rodent thyroid tumors are contradictory. The U.S. Food and Drug Administration (FDA) concluded that findings with drugs that lead to increased levels of thyroid-stimulating hormone (TSH) in rats are not relevant to humans, whereas the U.S. Environmental Protection Agency (US EPA) concluded that chemicals that produce rodent thyroid tumors may pose a carcinogenic hazard for humans although the thyroid of rodents appears to be more sensitive to a carcinogenic stimulus than that of humans. Meanwhile, based on the CLP Criteria of the European Chemicals Agency (ECHA), rodent thyroid tumors caused by the induction of uridine-diphosphate-glucuronosyl transferases (UDGT) were assessed as not relevant to humans. To clarify these discrepant positions, the function and regulation of the thyroid gland are described and the types of thyroid tumors and the causes of their development in humans and animals are examined. Based on these data and the evidence that so far, except radiation, no chemical is known to increase the incidence of thyroid tumors in humans, it is concluded that rodent thyroid tumors resulting from continuous stimulation of the thyroid gland by increased TSH levels are not relevant to humans. Consequently, compounds that induce such tumors do not warrant classification as carcinogenic.

[Metals and their compounds as contaminants in food : Arsenic, cadmium, lead and aluminum]. / Metalle und ihre Verbindungen als Kontaminanten in Lebensmitteln : Arsen, Cadmium, Blei und Aluminium.

Metals and their compounds are ubiquitously distributed in the environment, thus reaching plant and animal derived food. While actual exposure levels in Europe do not give rise to concern for acute toxicity, chronic toxicity of some metals and metalloids cannot be completely ruled out. Thus, in the case of inorganic arsenic, an elevated risk of carcinogenicity in different organs cannot be excluded even under actual dietary exposure conditions. In the case of cadmium, nephrotoxicity may be a particular problem for certain subgroups, such as children. Regarding lead, exposure levels have dropped considerably during the last two decades; nevertheless, developmental neurotoxicity may still be a problem during prenatal development and early childhood. Also in the case of aluminum current dietary exposure levels are close to the tolerable weekly intake (TWI) value derived by the European Food Safety Authority (EFSA). Taken together, for all four examples, further reductions in exposure levels are required.

Impact of cadmium on hOGG1 and APE1 as a function of the cellular p53 status.

The tumor suppressor protein p53, often called the guardian of the genome, is involved in important cellular processes, such as cell cycle control, apoptosis and DNA repair. With respect to BER, p53 might physically interact with and affect the transcription of different BER proteins such as hOGG1, APE1 or Polß. In studies in HCT116 p53(-/-) cells previously published, activity and mRNA expression of hOGG1 were found to be significantly decreased, while down-regulation of APE1 mRNA and protein levels in response to genotoxic stress were only described in HCT116 p53(+/+) cells, but not in the isogenic p53 knockout cell line. The predominantly indirect genotoxic carcinogen cadmium inhibits the BER pathway and potentially interferes with zinc binding proteins such as p53. Therefore, this study was accomplished to investigate whether p53 is involved in the cadmium-induced inhibition of BER activity. To address this issue we applied a non-radioactive cleavage test system based on a Cy5-labeled oligonucleotide. We present evidence that p53 is not essential for hOGG1 and APE1 gene expression as well as OGG and APE activity in unstressed HCT116 cells; however, it plays an important role in the cellular response to cadmium treatment. Here, a direct involvement of p53 was only observed with respect to APE1 gene expression contributing to an altered APE activity, while OGG activity was presumably affected indirectly due to a stronger accumulation of cadmium in HCT116 p53(+/+) cells. In summary, p53 indeed affects the BER pathway directly and indirectly in response to cadmium treatment.

Identification through microarray gene expression analysis of cellular responses to benzo(a)pyrene and its diol-epoxide that are dependent or independent of p53.

Human colon carcinoma cells (HCT116) differing in p53 status were exposed to benzo(a)pyrene (BaP) or anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) and their gene expression responses compared by complementary DNA microarray technology. Exposure of cells to BPDE for up to 24 h resulted in gene expression profiles more distinguishable by duration of exposure than by p53 status, although a subset of genes were identified that had significantly different expression in p53 wild-type (WT) cells relative to p53-null cells. Apoptotic signalling genes were up-regulated in p53-WT cells but not in p53-null cells and, consistent with this, reduced viability and caspase activity were also p53 dependent. BPDE modulated cell cycle and histone genes in both cell lines and, in agreement with this, both cell lines accumulated in S phase. In p53-WT cells, G(2) arrest was also evident, which was associated with accumulation of CDKN1A. Regardless of p53 status, exposure to BaP for up to 48 h had subtle effects on gene transcription and had no influence on cell viability or cell cycle. Interestingly, DNA adduct formation after BaP, but not BPDE, exposure was p53 dependent with 10-fold lower levels detected in p53-null cells. Other cell lines were investigated for BaP-DNA adduct formation and in these the effect of p53 knockdown was also to reduce adduct formation. Taken together, these results give further insight into the role of p53 in the response of human cells to BaP and BPDE and suggest that loss of this tumour suppressor can influence the metabolic activation of BaP.

Impact of copper on the induction and repair of oxidative DNA damage, poly(ADP-ribosyl)ation and PARP-1 activity.

Copper is an essential trace element involved, among other functions, in enzymatic antioxidative defense systems. However, nonprotein bound copper ions have been shown to generate reactive oxygen species. To gain insight into the discrepancy between the protective properties of copper on the one hand and its toxicity on the other hand, we examined the genotoxic effects of CuSO(4) in cultured human cells. Here we report that copper, at cytotoxic concentrations, induces oxidative DNA base modifications and DNA strand breaks. However, at lower noncytotoxic concentrations, copper inhibits the repair of oxidative DNA damage induced by visible light. As a first mechanistic hint, inhibition of H(2)O(2)-induced poly(ADP-ribosyl)ation was identified in cultured cells and further experiments demonstrated a strong inhibition of the activity of isolated poly(ADP-ribose)polymerase-1 (PARP-1) by copper. Bioavailability studies of copper showed a dose-dependent uptake in cells and pointed out the relevance of the applied concentrations. Taken together, the results indicate that copper, under conditions of either disturbed homeostasis or overload due to high exposure, exerts defined genotoxic effects. Hence, a balance needs to be maintained to ensure sufficient uptake and to prevent overload.