RESUMO
BACKGROUND: Global concern about vitamin D deficiency has fuelled debates on photoprotection and the importance of solar exposure to meet vitamin D requirements. OBJECTIVES: To review the published evidence to reach a consensus on the influence of photoprotection by sunscreens on vitamin D status, considering other relevant factors. METHODS: An international panel of 13 experts in endocrinology, dermatology, photobiology, epidemiology and biological anthropology reviewed the literature prior to a 1-day meeting in June 2017, during which the evidence was discussed. Methods of assessment and determining factors of vitamin D status, and public health perspectives were examined and consequences of sun exposure and the effects of photoprotection were assessed. RESULTS: A serum level of ≥ 50 nmol L-1 25(OH)D is a target for all individuals. Broad-spectrum sunscreens that prevent erythema are unlikely to compromise vitamin D status in healthy populations. Vitamin D screening should be restricted to those at risk of hypovitaminosis, such as patients with photosensitivity disorders, who require rigorous photoprotection. Screening and supplementation are advised for this group. CONCLUSIONS: Sunscreen use for daily and recreational photoprotection does not compromise vitamin D synthesis, even when applied under optimal conditions. What's already known about this topic? Knowledge of the relationship between solar exposure behaviour, sunscreen use and vitamin D is important for public health but there is confusion about optimal vitamin D status and the safest way to achieve this. Practical recommendations on the potential impact of daily and/or recreational sunscreens on vitamin D status are lacking for healthy people. What does this study add? Judicious use of daily broad-spectrum sunscreens with high ultraviolet (UV) A protection will not compromise vitamin D status in healthy people. However, photoprotection strategies for patients with photosensitivity disorders that include high sun-protection factor sunscreens with high UVA protection, along with protective clothing and shade-seeking behaviour are likely to compromise vitamin D status. Screening for vitamin D status and supplementation are recommended in patients with photosensitivity disorders.
Assuntos
Medicina Baseada em Evidências/normas , Neoplasias Cutâneas/prevenção & controle , Luz Solar/efeitos adversos , Protetores Solares/efeitos adversos , Deficiência de Vitamina D/prevenção & controle , Vitamina D/sangue , Consenso , Saúde Global/normas , Humanos , Programas de Rastreamento/normas , Recreação , Valores de Referência , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/efeitos da radiação , Neoplasias Cutâneas/etiologia , Fator de Proteção Solar , Protetores Solares/administração & dosagem , Protetores Solares/química , Raios Ultravioleta/efeitos adversos , Vitamina D/administração & dosagem , Vitamina D/metabolismo , Deficiência de Vitamina D/sangue , Deficiência de Vitamina D/diagnóstico , Deficiência de Vitamina D/epidemiologiaRESUMO
Skin is particularly exposed to oxidative stress, either from environmental insults such as sunlight or pollution or as a consequence of specific impairments in antioxidant status resulting from pathologies or aging. Traditionally, antioxidant products are exogenously provided to neutralize pro-oxidant species. However, another approach based on stimulation of endogenous antioxidant defense pathways is more original. Resveratrol (RSV) was reported to display such a behavior in various tissues, but data about the mechanisms of action in skin are scarce. We show here that, in primary culture of normal human keratinocytes (NHKs) or in full-thickness reconstructed human skin, RSV activated the Nrf2 pathway at nontoxic doses, from 20 µM up to 100µM. Among the Nrf2 downstream genes, glutamylcysteinyl ligase and glutathione peroxidase-2 were induced at the mRNA and protein levels. In parallel, a significant increase in glutathione content, assessed by LC/MS analysis, was observed in both models. Nrf2 gene silencing experiments performed in NHKs confirmed that Nrf2 was involved in RSV-induced modulation of cellular antioxidant status, in part by increasing cellular glutathione content. Finally, improvement of endogenous defenses induced in RSV-pretreated reconstructed skin ensured protection against the toxic oxidative effects of cumene hydroperoxide (CHP). In fact after RSV pretreatment, in response to CHP stress, glutathione content did not decrease as in unprotected samples. Cellular alterations at the dermal-epidermal junction were clearly prevented. Together, these complementary experiments demonstrated the beneficial effects of RSV on skin, beyond its direct antioxidant properties, by upregulation of a cutaneous endogenous antioxidant pathway.
Assuntos
Antioxidantes/farmacologia , Glutationa/metabolismo , Queratinócitos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Absorção Cutânea/efeitos dos fármacos , Estilbenos/farmacologia , Western Blotting , Células Cultivadas , Cromatografia Líquida , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Fator 2 Relacionado a NF-E2/genética , Transporte Proteico , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Resveratrol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In vitro and in vivo studies provide a body of evidence that adequate protection of the skin against ultraviolet (UV)-induced damage requires photostable broad-spectrum sunscreens with a proper level of UVA protection. UVA alone and UV solar simulated radiation (SSR) induce DNA lesions in keratinocytes and melanocytes as reflected by the comet assay and p53 accumulation. UVA and SSR impair the immune system as shown by significant alteration of Langerhans cells and inhibition of contact hypersensitivity response to chemical allergens and delayed-type hypersensitivity response to recall antigens. Any of these detrimental effects is more efficiently prevented by sunscreens with a higher level of protection in the UVA range. The involvement of UVA (fibroblast alteration, increased metalloproteinase expression) and the pivotal need for well-balanced UVA/UVB sunscreens were further demonstrated using reconstructed three-dimensional skin models.
Assuntos
Sistema Imunitário/efeitos da radiação , Queratinócitos/efeitos da radiação , Melanócitos/efeitos da radiação , Queimadura Solar/prevenção & controle , Protetores Solares/farmacologia , Proteína Supressora de Tumor p53/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Dermatite de Contato/etiologia , Humanos , Técnicas In Vitro , Envelhecimento da Pele/efeitos da radiação , Queimadura Solar/imunologia , Protetores Solares/efeitos adversosRESUMO
BACKGROUND: Acute as well as chronic sun exposure induces biologically damaging effects in skin including photoageing and cancer. Ultraviolet (UV)A radiation is involved in this process; it is therefore important that sunscreen products provide efficient and stable protection in this range of wavelengths. OBJECTIVES: This study based on in vitro approaches was performed to demonstrate that photostability is an essential requirement to protect against UVA-induced genetic and dermal alterations. METHODS: The protection afforded by two sunscreen products, differing with regard to their photostability, was studied using biological markers related to the genotoxic or photoageing impact of UVA or simulated solar UV radiation (UV-SSR). Comet assay was used to assess direct DNA breakage, photo-oxidized purines and lomefloxacin-induced DNA breaks in nuclei of normal human keratinocytes in culture. In similar conditions, detection of p53 accumulation was performed. The use of reconstructed skin in vitro allowed us to use a three-dimensional model to analyse the dermal and epidermal damage induced by UVA or UV-SSR exposure. Abnormal morphological features of the tissue as well as fibroblast alterations and matrix metalloproteinase-1 release induced by UV exposure have been studied after topical application of products on the skin surface. RESULTS: The results showed that the photostable product afforded better protection with regard to all the criteria studied, compared with the photounstable product. CONCLUSIONS: These data demonstrate that the loss of absorbing efficiency within the UVA wavelength domain due to photoinstability may have detrimental consequences on cell function and lead to impairments that have been implicated in genotoxic events as well as in the photoageing process.
Assuntos
Dano ao DNA/efeitos dos fármacos , Envelhecimento da Pele/efeitos dos fármacos , Protetores Solares/química , Raios Ultravioleta , Técnicas de Cultura de Células , Ensaio Cometa , Relação Dose-Resposta à Radiação , Estabilidade de Medicamentos , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Pele/efeitos da radiação , Envelhecimento da Pele/efeitos da radiação , Pele Artificial , Protetores Solares/farmacologia , Protetores Solares/efeitos da radiação , Técnicas de Cultura de Tecidos , Proteína Supressora de Tumor p53/metabolismoRESUMO
Today's lifestyle is often associated with frequent exposure to sunlight, but some xenobiotics used in drugs, cosmetics or food chemicals can produce adverse biological effects when irradiated. In particular, they can increase the risk of photogenotoxicity already due to UV radiation itself. There is thus a need to design appropriate approaches in order to obtain relevant data at the molecular and cellular level in this field. For ethical and practical reasons, in vitro models can be very convenient at least for first evaluation tests. Here, we propose a strategy based on complementary experiments to study the photogenotoxic potential of a compound. The fluoroquinolones BAYy3118 and lomefloxacin were used as standards to demonstrate the performance of each test: photoinduced interaction with supercoiled circular DNA, photomutagenicity in the yeast Saccharomyces cerevisae, induction of DNA photodamage in cultured human skin cells as revealed by comet assay, and finally induction of specific phototoxic stress responses such as p53 activation or melanogenesis stimulation. Such a strategy should help to ensure the safety of products likely to undergo environmental sunlight exposure.
Assuntos
Anti-Infecciosos/farmacologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , DNA Super-Helicoidal/efeitos da radiação , Fluoroquinolonas , Testes de Mutagenicidade/métodos , Quinolonas/farmacologia , Raios Ultravioleta/efeitos adversos , Anti-Infecciosos/toxicidade , Linhagem Celular , Ensaio Cometa , Humanos , Técnicas In Vitro , Melanócitos/efeitos da radiação , Monofenol Mono-Oxigenase/efeitos da radiação , Fotoquímica , Pigmentação/efeitos da radiação , Quinolonas/toxicidade , Saccharomyces cerevisiae/genética , Pele/efeitos da radiação , Proteína Supressora de Tumor p53/efeitos da radiaçãoRESUMO
Fluoroquinolones are antibiotics with a potential clinical side effect of phototoxicity and some are suspected to enhance UVA-induced tumorigenesis. The present study was designed to evaluate the recombinogenic and mutagenic potential of two highly photoreactive compounds, lomefloxacin and BAYy3118 when exposed to complete UVA (320-400 nm). In order to possibly increase the sensitivity of the test, we used a diploid mutant (D7-rad3) deficient in nucleotide excision repair and deriving from the tester strain D7 of the yeast Saccharomyces cerevisae. In agreement with previous reports, lomefloxacin had no effect in this system. Moreover, BAYy3118 was highly photocytotoxic and genotoxic especially when yeast cells were incubated in its presence in the dark before exposure to UVA radiation. Both fluoroquinolones were comparable in their ability to photo-induce DNA strand breaks or oxidative damage to purines and pyrimidines in supercoiled plasmid DNA, but agarose gel electrophoresis showed that BAYy3118 photoproducts could tightly interact with supercoiled plasmid DNA while lomefloxacin ones only induced strand breaks. These data suggest that phototoxicity of BAYy3118 was the result of a multistep mechanism: first, local photo oxidative stress is induced and secondly some of the photoproducts exerted genotoxic effects. This work also shows that very simple and complementary in vitro approaches can be very informative in the understanding of drug-induced phototoxicity.
Assuntos
Anti-Infecciosos/toxicidade , DNA Super-Helicoidal/efeitos dos fármacos , Fluoroquinolonas , Fármacos Fotossensibilizantes/toxicidade , Plasmídeos/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Adenosina Trifosfatases/genética , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , DNA Helicases/genética , DNA Super-Helicoidal/efeitos da radiação , Diploide , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Testes de Mutagenicidade , Mutação , Plasmídeos/efeitos da radiação , Quinolonas/toxicidade , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae , Fatores de Tempo , Raios UltravioletaRESUMO
The fluoroquinolone antibiotic, lomefloxacin, is phototoxic in human skin exposed to UVA radiation, photosensitises DNA strand breaks and pyrimidine dimers in human keratinocytes in vitro, and is phototumorigenic in mouse skin. The p53 tumour suppressor protein is activated by a variety of cellular insults including UV radiation, to become a transcription factor for downstream markers such as the cyclin-kinase inhibitor p21CIP1/WAF1 or cause caspase transactivation which cleaves poly ADP ribose polymerase (PARP) as an early step in apoptosis. We have investigated these molecular defence responses in human skin cells treated with lomefloxacin and UVA radiation in vitro. Western blots revealed that lomefloxacin photosensitised the stabilisation of p53 protein in human fibroblasts. Lomefloxacin also photosensitised p53 transcriptional activity in amelanotic melanoma cells expressing wild-type p53 and stably transfected with a construct containing a beta-galactosidase reporter gene downstream from a p53 consensus binding sequence. Neither photosensitised production of H2O2 nor the resultant DNA strand breaks, appeared to be involved in this effect. Interestingly, p21CIP1/WAFI protein was upregulated by lomefloxacin in the dark by a p53-independent mechanism. Lomefloxacin also photosensitised the degradation of nuclear PARP, suggestive of caspase mediated, early apoptotic events.
Assuntos
Anti-Infecciosos/farmacologia , Fluoroquinolonas , Fármacos Fotossensibilizantes/farmacologia , Quinolonas/farmacologia , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Anti-Infecciosos/química , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Dano ao DNA , Humanos , Peróxido de Hidrogênio/metabolismo , Estrutura Molecular , Fármacos Fotossensibilizantes/química , Quinolonas/química , Pele/citologia , Proteína Supressora de Tumor p53/genéticaRESUMO
The induction of DNA breaks by UVA (320-400 nm) in the nucleus of normal human melanocytes in culture was investigated using single cell gel electrophoresis, also called the comet assay. Endogenous pigment and/or melanin-related molecules were found to enhance DNA breakage: comets were more intense in melanocytes than in fibroblasts, in cells with high melanin content or after stimulation of melanogenesis by supplying tyrosine in the culture medium. After UVA doses where strong comets were observed, neither cytotoxicity nor stimulation of tyrosinase activity were detected. However, the accumulation of p53 protein suggested that cells reacted to genotoxic stress under these experimental conditions. The same approach was used to compare two sunscreens with identical sun protection factors but different UVA protection factors. The results presented in this paper suggest that human melanocytes may be used as a target cell to evidence broadspectrum photoprotection. Moreover, these data appear to be helpful in getting a better understanding of the role of sunlight in the initiating steps of melanocyte transformation.
Assuntos
Melanócitos/efeitos da radiação , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos da radiação , Dano ao DNA , Humanos , Melaninas/metabolismo , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Fotobiologia , Protetores Solares/farmacologia , Raios Ultravioleta/efeitos adversosRESUMO
Skin cancers are among the most common human cancers and have an increasing incidence. The ultraviolet radiation components of sunlight play a major role in skin tumor induction and development. Cellular DNA has been identified as a target for most of the biological effects of UV, and the induction of photodamage is considered as the initiating step of photocarcinogenesis. Thus, effective photoprotection of DNA against harmful overex-posure to solar UV is a critical issue. The efficiency of a sunscreen is usually tested with respect to its ability to prevent skin erythema, but conceivably, more data are required at the molecular and cellular level in order to ascertain protection against photocarcinogenic risk. In the present study, we define a strategy based on the use of various in vitro models and solar-simulated light to evaluate photodamage and photoprotection: -Supercoiled circular plasmid DNA for detection of structural alterations. -The yeast Saccharomyces cerevisiae to evaluate cytotoxicity and genotoxicity. -The single-cell gel electrophoresis or comet assay to determine DNA damage and DNA repair in human keratinocytes. -p53 expression as a hallmark for genotoxic stress. -Induction of pigmentation in human melanocytes. In conditions where light source, spectrum and control of radiation delivery were precisely defined, we have demonstrated that the wide spectrum UVA sunscreen Mexoryl SX protects from the cytotoxicity and genotoxicity of solar UV.
Assuntos
Cânfora/análogos & derivados , Dermatite Fototóxica/etiologia , Dermatite Fototóxica/prevenção & controle , Mesilatos/administração & dosagem , Pele/efeitos da radiação , Protetores Solares/administração & dosagem , Raios Ultravioleta/efeitos adversos , Western Blotting , Canfanos , Cânfora/administração & dosagem , Morte Celular/efeitos da radiação , Sobrevivência Celular , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/efeitos da radiação , DNA Mitocondrial/análise , DNA Mitocondrial/efeitos da radiação , Dermatite Fototóxica/diagnóstico , Eletroforese , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/efeitos da radiação , Mitose/efeitos da radiação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Sensibilidade e Especificidade , Pele/citologia , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/prevenção & controle , Ácidos Sulfônicos , Proteína Supressora de Tumor p53/análiseRESUMO
N,N'-bis-(3,4,5-trimethoxybenzyl) ethylenediamine N,N,-diacetic acid dihydrochloride (OR10141) is a member of a recently described series of "oxidative stress activatable iron chelators." These chelators have a relatively low affinity for iron but can be site-specifically oxidized, in situations mimicking oxidative stress in vitro, into species with strong iron-binding capacity. It is hoped that this local activation process will minimise toxicity compared to strong iron chelators that may interfere with iron metabolism. The present paper describes the results of experiments aimed at characterising oxidative reactions between iron-OR10141 complexes and hydrogen peroxide. Incubation of ascorbate and hydrogen peroxide with the ferric chelate of OR10141 in neutral aqueous solution yields a purple solution with a chromophore at 560 nm, which is consistent with an o-hydroxylation of one of the trimethoxybenzyl rings. Oxidation of OR10141 also takes place, although more slowly, by incubating hydrogen peroxide with ferric OR10141 complex in the absence of reductant. HPLC analysis shows that OR10141 is consumed during the reaction and transformed principally into N-(2-hydroxy 3,4,5-trimethoxybenzyl) N'-(3,4,5-trimethoxybenzyl) ethylenediamine N,N'-diacetic acid. Minor products are also formed, some of which were identified by mass spectrometry. The protective effect of OR10141 in vitro against DNA single strand breaks, protein damage, and lipid peroxidation induced by Fenton chemistry suggests that this compound is able to compete for iron with biological molecules and, thus, that this strategy of protection against oxidative stress is feasible. In addition, preliminary results showing protective effects of OR10141 dimethyl ester against toxicity induced by hydrogen peroxide in cell culture are described. It is concluded that OR10141 and related prodrugs might be useful in vivo in chronic situations involving oxidative stress.
Assuntos
Ácido Edético/análogos & derivados , Quelantes de Ferro/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Ácido Ascórbico/química , Linhagem Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Dano ao DNA/efeitos dos fármacos , DNA de Cadeia Simples/efeitos dos fármacos , Ácido Edético/síntese química , Ácido Edético/farmacologia , Glucose Oxidase , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Oxirredução , EspectrofotometriaRESUMO
A concentration of H2O2 resulting in mode one killing of Escherichia coli is more toxic when exposure to the oxidant is performed in complete medium (K medium), as compared to a saline (M9 salts). Inorganic salts (MgSO4 and CaCl2), thiamine or glucose, when added separately, or combined, to M9 salts had no effect on the cytotoxic response to H2O2. In contrast, the lethality of the oxidant was highly dependent on the presence of the amino acids in the incubation medium. The addition of glucose further enhanced this response. Among the seventeen amino acids which are present in the complete amino acid mixture, only two, i.e. L-histidine and L-cystine, were found to increase the toxicity of H2O2. Again, glucose augmented this response. The effect of these amino acids on the growth inhibitory action of hydrogen peroxide was also tested in Chinese Hamster Ovary cells. It was found that L-histidine was capable of increasing the toxicity of the oxidant whereas all the other amino acids did not affect the toxicity of the oxidant. Glucose only slightly augmented this effect of L-histidine. DNA single strand breakage produced by H2O2 was increased by L-histidine and was not significantly modified by the other amino acids. DNA double strand breakage was also shown to occur in cells exposed to H2O2-L-histidine, and this effect was independent on the presence of glucose. These results demonstrate that the cytotoxic response of bacterial and mammalian cells to challenge with H2O2 is highly dependent on the composition of the extracellular milieu.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Aminoácidos/farmacologia , Células CHO/efeitos dos fármacos , Meios de Cultura/farmacologia , Escherichia coli/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Cistina/farmacologia , Dano ao DNA , Sinergismo Farmacológico , Glucose/farmacologia , Histidina/farmacologia , Oxirredução , Sais/farmacologiaRESUMO
The purpose of this work was the comparison of the conformational changes induced in the double helix by the adducts formed at d(GG) and d(AG) sites in the reaction between the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) and DNA. Two duplexes (20-mer) containing either a single d(A*G*) or a single d(G*G) adduct were studied by means of gel electrophoresis and artificial nuclease and chemical probes. It is shown that the d(G*G*) and the d(A*G*) adducts bend DNA similarly, but at the nucleotide level they distort differently the double helix. We suggest that the weaker interactions between platinated A residues and the other nucleotides, as compared to the interactions between platinated G residues and the other nucleotides, are largely responsible for the differences in the distortions induced in DNA by the d(A*G) and d(G*G*) adducts. This suggestion is supported by the study of the distortions induced in duplexes by the d(G*G*) adducts, one of the platinated G residues being paired with a T residue.
Assuntos
Adenina/análise , Cisplatino/farmacologia , Dano ao DNA , DNA/efeitos dos fármacos , Guanina/análise , Conformação de Ácido Nucleico , Sequência de Bases , DNA/análise , Eletroforese , Dados de Sequência Molecular , Ácidos Nucleicos Heteroduplexes/efeitos dos fármacos , Sondas de OligonucleotídeosRESUMO
The reactivity of nucleic acids in various conformations and two isosteric chemical carcinogens 2-N,N-acetoxyacetylaminofluorene (N-AcO-AAF) and 3-N,N-acetoxyacetylamino-4,6-dimethyldipyrido [1,2-a:3',2'-d] imidazole (N-AcO-AGlu-P-3) have been studied. Both carcinogens bind covalently to poly(dG-dC).poly(dG-dC) (B form) and to poly(dG-br5C).poly(dG-br5dC) (Z form). They also bind covalently to (dC-dG)16 and to (dG-dT)15 sequences inserted in plasmids when the inserts are in the B form but they do not bind to the inserts in the Z form. The reactivity of guanine residues at the B-Z junctions depends upon the superhelical density of the plasmids and upon the base sequences at the junction. The distribution of AGlu-P-3 modified guanines in a restriction fragment of pBR322 is not uniform and is different from that of AAF-modified guanines. The conclusion is that N-AcO-Glu-P-3 as N-AcO-AAF can probe at the nucleotide level the polymorphism of DNA. On the other hand, the non-reactivity of both chemical carcinogens and Z-DNA and the hyperreactivity of some junctions might have some importance in the understanding of chemical carcinogenesis.