Polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin induce intrachromosomal recombination in vitro and in vivo

Polychlorinated aromatic hydrocarbons such as polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are extremely stable and widely distributed environmental pollutants. These chemicals are animal carcinogens and probable human carcinogens, and TCDD is possibly one of the most potent toxins ever evaluated by the United States Environmental Protection Agency. Polychlorinated aromatic hydrocarbons score negatively in most genotoxicity assays, including the Ames (Salmonella) assay. Although their mechanism of toxicity is not well understood, they induce aryl hydrocarbon (AH) hydroxylases and bind to the AH receptor, which is believed to mediate toxicity. Here, we determine effects of polychlorinated aromatic hydrocarbons in genotoxicity assays that score for DNA deletions by intrachromosomal recombination in vivo and in vitro. In this study, TCDD, Aroclor 1221, and Aroclor 1260 induced deletions in vivo in the mouse embryo; Aroclor 1221 and Aroclor 1260 induced deletions in yeast. We also show that the induced deletion events did not correlate with induction of AH hydroxylase. None of the tested compounds induced CYP1A-associated ethoxyresorufin-O-deethylase activity in mouse embryos or in vitro. These results clearly demonstrate a genotoxic activity of polychlorinated aromatic hydrocarbons in vitro and in vivo, which is independent of induction of cytochrome P450 activity. Because genetic instability and deletions may be mechanistically involved in carcinogenesis, these results may encourage further research to determine whether such genotoxic mechanisms may be useful for cancer risk assessment of polychlorinated aromatic hydrocarbons.     

Tumors of the oral cavity, oropharynx and salivary glands. Role of TC and MRI in neoplasm staging

The 32P-postlabelling assay is one of the most sensitive methods for detection of DNA adducts induced by exposure to genotoxic chemicals. Under optimal conditions, detection limits of one adduct per 10(9)-10(10) nucleotides have been reported. This sensitivity now allows monitoring of occupational and even environmental exposure of humans to certain classes of chemicals, mainly polycyclic aromatic hydrocarbons (PAH). Despite its widespread use, 3P-postlabelling is still not a standardized method. Rigorous interlaboratory comparisons are scarce, and those that have been undertaken often show rather different results, both in relative and in absolute values, for the amounts of DNA adducts in the same samples. Furthermore, the optimization of many steps in the procedure has still not been given adequate attention. This paper deals with some technical aspects of detection of PAH-DNA adducts by 32P-postlabelling, in particular with assay calibration and adduct quantification. For this purpose, benzo[a]pyrene (BP)-modified DNA standards were prepared, the adduct contents of which were determined by use of an independent fluorometric method, viz. synchronous fluorescence spectrophotometry (SFS). These BP-DNA standards are processed along with the test samples throughout the entire 32P-postlabelling procedure, from the enzymic digestion up to and including the determination of radioactivity in adduct spots on the chromatogram. As such, these reference samples can be considered as external standards for inter-assay calibration. This method for adduct quantification was compared with the commonly used relative adduct labelling (RAL) and comparative dAMP labelling, which appeared to give rise to an underestimation of adduct levels. The method was applied in a biomonitoring study among workers in a carbon-electrode manufacturing plant, exposed to PAH. Although DNA adduct levels in peripheral blood lymphocytes of exposed workers, as determined by 32P-postlabelling, were not significantly different from those of controls, a significant difference was seen when smokers and non-smokers were compared.     

The burnout of nursing staff working with challenging behaviour clients in hospital-based bungalows and a community unit

The increasing presence of genotoxic chemicals in the aquatic environment has led to the development of both in vivo and in vitro assays for target species. The fish population represents an important level of aquatic ecosystems that can be threatened by increased environmental pollution. The authors have studied the DNA pattern of the RTG-2 fish cell line, a fibroblast-like cell line, derived from rainbow trout (Oncorhynchus mikyss), to use this cell line as an in vitro system to study genotoxicity by means of random amplified polymorphic DNA primers (RAPDs). A constant pattern in the DNA band is essential when an organism or cell line is used to detect DNA alterations produced by genotoxic environmental chemicals. DNA fingerprints with RAPDs were obtained for RTG-2 by testing 26 single and 70 pairwise combinations of primers. Different methods of DNA extraction (chelating resin, salting out, and phenolization), the influence of spectrometric measures at 320 nm in the 260/280 quotient to quantify DNA extracts, genomic DNA and primer concentrations, annealing temperatures, and cell line passage were studied in the cell line characterization. RAPD products were identified by agarose gel electrophoresis. The good results obtained should allow the use of this system as a possible tool for detection of the genotoxicity of aquatic pollutants.     

Comparison of immunoaffinity chromatography enrichment and nuclease P1 procedures for 32P-postlabelling analysis of PAH-DNA adducts

32P-postlabelling analysis for detecting DNA adducts formed by polycyclic aromatic compounds is one of the most widely used techniques for assessing genotoxicity associated with these compounds. In cases where the formation of adducts is extremely low, a crucial step in the analysis is an enrichment procedure for adducts prior to the radiolabelling step. The nuclease P1 enhancement procedure is the most established and frequently used of these methods. An immunoaffinity procedure developed for class specific recognition for polycyclic aromatic hydrocarbon (PAH)-DNA adducts has therefore been compared with the nuclease P1 method for a range of DNA adducts formed by PAHs. The evaluation was carried out with skin DNA from mice treated topically with benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 5-methylchrysene or chrysene. The immobilised antibody had the highest affinity for adducts structurally similar to the BPDE-I-deoxyguanosine adduct ([+/-]-N2-(7r,8t,9r-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-1 0t-yl)-2'-deoxyguanosine) against which the antibody had been raised. Of the PAH-modified DNAs evaluated, the maximum adduct recovery was obtained for DNA containing the BPDE I-deoxyguanosine adduct. With DMBA-modified DNA, the profiles of adducts recovered from the column were similar when the column material was treated either with a digest of DMBA-modified DNA or with 32P-labelled DMBA adducts. I-compounds (endogenous adducts in tissue DNA of unexposed animals), which had similar chromatographic properties to PAH-DNA adducts, were not enriched by the immunoaffinity procedure. Compared to the simple nuclease P1 enhancement procedure, the immunoaffinity methods were lengthier and more labour intensive. Advantages of the immunoaffinity procedure include: specificity, allowing the selective detection of a certain class of adducts: efficient adduct enrichment, providing a viable alternative to other enrichment procedures; adequate sensitivity for model studies and the potential to purify adducts for further characterisation. However, as a general screen for detecting the formation of DNA adducts, the nuclease P1 procedure was viewed as the initial method of choice since it was capable of detecting a wider range of PAH-DNA adducts.     

Aromatic DNA adducts in larynx biopsies and leukocytes

Larynx cancer is strongly associated with tobacco smoking. The objective of this work was an analysis of aromatic DNA adducts in tumour and non-tumour larynx cells by means of the 32P-postlabelling method. Peripheral blood leukocytes were used as a reference tissue. The presence of aromatic DNA adducts was demonstrated in all the studied tissues obtained after surgery of larynx tumours. The highest level of DNA adducts was found in larynx tumour cells, followed by non-tumour larynx cells, which exceeded that found in leukocytes almost 2.5 times. Large interindividual differences were detected between subjects. The adduct level in tumour/non-tumour correlated only moderately. However a high correlation was found between the level of DNA adducts in larynx (tumour and non-tumour) cells and that in leukocytes.     

Comparison of total DNA adduct levels induced in mouse tissues and human skin by mainstream and sidestream cigarette smoke condensates

Cigarette smoke condensates (CSCs) of both mainstream (MS) and sidestream (SS) smoke were used to treat mice topically in equivalent amounts. Human skin maintained in short-term culture was also treated with the condensates. DNA adducts, induced by the CSCs and detected by the nuclease P1 method of 32P-postlabelling, were quantified in a number of murine tissues and in the human skin DNA. In the five mouse tissues studied both MS-CSC and SS-CSC produced characteristic diagonal radioactive zones on TLC, indicative of the formation of multiple DNA adducts. In three tissues (skin, lung and kidney), SS-CSC induced greater total adduct levels than MS-CSC (statistically significant in skin and kidney, p < 0.05). However, greater adduct levels induced by MS-CSC were recorded for heart and bladder DNA (not statistically significant). Similar results to those found in mouse skin were obtained with human skin; SS-CSC induced a approximately 2-fold greater level of DNA adducts than MS-CSC (p < 0.05). Incubation of DNA directly with condensates in vitro demonstrated that DNA adducts could be formed without an exogenous metabolizing system. This direct interaction of condensates with DNA occurred at similar levels for both MS- and SS-CSC, although inclusion of an oxygen radical-generating system enhanced the SS-CSC binding to a greater extent than that of the MS-CSC.     

Monophosphate 32P-postlabeling assay of DNA adducts from 1,2:3,4-diepoxybutane, the most genotoxic metabolite of 1,3-butadiene: in vitro methodological studies and in vivo dosimetry

Among the main DNA-reactive metabolites of 1,3-butadiene (BD), both 1,2:3,4-butadiene diepoxide (BDE) and 1,2-epoxy-3-butene (BME) have been reported in mice and rats exposed to BD, but blood and tissue levels of these metabolites are much higher in mice than in rats under similar exposure conditions. BDE, being more reactive and genotoxic than BME, is thought to be responsible for the greater susceptibility of mice to BD carcinogenicity. While BDE is a DNA-alkylating agent and some BDE adducts have been characterized, no sufficiently sensitive method has been reported for studying BDE-DNA binding in vivo. In the present investigation, a modified dinucleotide/monophosphate version of the 32P-postlabeling assay was applied to detect BDE-DNA adducts, which were prepared by reacting BDE with calf thymus DNA or deoxyribooligonucleotides [(AC)10, (AG)10, (CCT)7 and (GGT)7] in vitro or with skin DNA of mice in vivo upon topical treatment. Optimal resolution by 2-D PEI-cellulose TLC of the highly polar 5'-monophosphate adducts was achieved at +4 degrees C using 0.3 M LiCI (DI) and 0.4 M NaCl, 0.04 M H3BO3, pH 7.6 (D2). The profiles of the 32P-postlabeled adducts were similar for calf thymus and skin DNA, with 3 major spots being detected. Adducts obtained in in vitro and in vivo experiments were compared by re- and cochromatography in 4 or 5 different solvents, and these experiments provided evidence that corresponding BDE adducts, for the most part, were identical and represented adenine derivatives. Guanine adducts were not detected by this method although literature data indicate their formation. Quantitatively, the assay responded linearly to adduct concentration, as shown in an experiment where BDE-modified skin DNA was serially diluted up to 81-fold with control DNA. The limit of detection was approximately 1 adduct in 10(8) normal nucleotides. Further, in an in vivo dosimetry study, skin DNA from groups of 8 individual mice treated with different doses of BDE (1.9, 5.7, 17, 51 and 153 mumol/mouse) for 3 days exhibited a linear relationship (r > or = 0.992) between adduct levels and dose. The results suggest that the 32P-postlabeling assay described herein will have utility in mechanistic studies and biomonitoring of DNA adduct formation from BDE and possibly other polar epoxides.     

Effect of various marine lipids on transdermal drug delivery--in vitro evaluation

The effects of several marine lipids on the penetration of hydrocortisone and nitroglycerin through excised hairless mouse skin have been studied. Fatty acid extracts obtained by hydrolysis of Portuguese dog-fish-liver-oil or by hydrolysis of cod-liver-oil were shown to be effective skin penetration enhancers. Phospholipid obtained from squid was also shown to be effective enhancer. However, the enhancing effect of the marine products could generally be associated with their content of free unsaturated fatty acids. The fatty acid extract obtained from cod-liver-oil caused insignificant skin irritation when incorporated into an ointment base and applied to human skin.     

Series: current issues in mutagenesis and carcinogenesis, No. 65. The genotoxicity and carcinogenicity of paracetamol: a regulatory (re)view

The publication of several studies reporting genotoxic effects of paracetamol, one of the world's most popular over-the-counter drugs, has raised the question of regulatory action. Paracetamol does not cause gene mutations, either in bacteria or in mammalian cells. There are, however, published data giving clear evidence that paracetamol causes chromosomal damage in vitro in mammalian cells at high concentrations and indicating that similar effects occur in vivo at high dosages. Available data point to three possible mechanisms of paracetamol-induced genotoxicity: (1) inhibition of ribonucleotide reductase; (2) increase in cytosolic and intranuclear Ca2+ levels; (3) DNA damage caused by NAPQI after glutathione depletion. All mechanisms involve dose thresholds. Studies of the relationship between genotoxicity and toxic effects in the rat (induction of micronuclei in rat bone marrow including dose-response relationship, biotransformation of paracetamol at different dosages, concomitant toxicity and biochemical markers) have recently been completed. These studies, which employed doses ranging from the dose resulting in human therapeutic peak plasma levels to highly toxic doses, give convincing evidence that genotoxic effects of paracetamol appear only at dosages inducing pronounced liver and bone marrow toxicity and that the threshold level for genotoxicity is not reached at therapeutic dosage. Reliable studies on the ability of paracetamol to affect germ cell DNA are not available. However, based on the amount of drug likely to reach germ cells and the evidence of thresholds, paracetamol is not expected to cause heritable damage in man. Various old and poorly designed long-term studies of paracetamol in the mouse and rat have given equivocal results. A few of these studies showed increased incidence of liver and bladder tumours at hepatotoxic doses. National Toxicology Program (U.S.A.) feeding studies have shown that paracetamol is non-carcinogenic when given at non-hepatotoxic doses up to 300 mg/kg/d to the rat and up to 1000 mg/kg/d to the mouse. Taking into account the knowledge of the hepatotoxicity and metabolism of paracetamol and the existence of thresholds for its genotoxicity, the animal studies do not indicate a carcinogenic potential at non-hepatotoxic dose levels. Based on this updated assessment of the genotoxicity and carcinogenicity of paracetamol, it is concluded that there is no need for regulatory action.     

Effect of energy shortage and oxidative stress on amyloid precursor protein metabolism in COS cells

The standard method for assessing the carcinogenicity of lubricating oil base stocks is the mouse skin-painting bioassay. This assay has the advantage of directly measuring the endpoint of interest, dermal carcinogenicity, but has the drawback of being time-consuming and expensive. For this reason, a variety of biological and chemical assays have been developed as predictive alternatives to the in vivo assay. This publication describes the application of three such methods to the assessment of carcinogenic potential of hydrotreated, re-refined oils: the modified Ames test, the analytical determination of 3-7-ring polycyclic aromatic compound content and the 32P-postlabeling assay for DNA adduct induction.     

Genotoxic activity of 1-chloromethylpyrene in stomach epithelium in vivo: insensitivity of the stomach scintillation UDS assay

An acknowledged weakness of current testing programmes for genotoxic hazard has been the potential insensitivity of the established mouse bone marrow micronucleus test and rat liver unscheduled DNA synthesis (UDS) assays to direct-acting or short-lived mutagens, which may be consumed at the site of initial contact. In such cases, in vivo test systems sampling tissues such as the skin or the stomach would provide valuable data. To test these principles a stomach UDS assay was evaluated using the potent locally active mutagen 1-chloromethylpyrene (1-CMP). Contrary to expectations, no UDS response was observed 16 h following 1-CMP dosage by oral gavage. To confirm the integrity of the 1-CMP used for the stomach UDS assay, a sample of the stored chemical was re-evaluated in vitro and shown to be still strongly positive in the Ames assay and to have alkylating activity at least 15 min after incubation at stomach acid pH. No UDS response was observed when test dose levels were reduced or when earlier sampling times were used. Other genotoxic endpoints were examined in stomach. 32P-Postlabelling analysis revealed high levels of adduct formation in gastric DNA. An assay utilizing electrophoresis of DNA (the comet assay) showed the occurrence of DNA damage following dosing with 1-CMP in vivo. These positive results confirmed that 1-CMP should be regarded as a potential in vivo genotoxin. The failure to detect a UDS response to 1-CMP in stomach was investigated; a strong UDS response was observed in an in vitro hepatocyte UDS assay of 1-CMP indicating that the rat was capable of repairing 1-CMP-derived DNA adducts. Pretreatment of rats with hydroxyurea depressed the level of incorporation of thymidine into DNA both in negative and positive [methyl-N-nitrosoguanidine (MNNG)] controls. The results of these studies indicated that the protease digestion method employed did not selectively or efficiently sample those cells with any UDS response to 1-CMP or MNNG, and the activity seen for the latter was most likely due to the presence of S phase cells within the digests. As a result of the finding that UDS responses were not demonstrated for the potent direct-acting mutagens 1-CMP and MNNG, the protease digestion/scintillation method for stomach UDS does not appear to have general value in a screening programme for locally active genotoxic agents.     

S-oxygenation of thiourea results in the formation of genotoxic products

Thiourea (TU) is a thyroid carcinogen which has previously been shown to cause genotoxicity in various test systems in vitro and in vivo. The mechanism underlying these effects has not yet been elucidated. The present study addressed the question of whether the formation of oxidized products of TU might be involved in genotoxicity. Chemical oxidation of [14C]TU with hydrogen peroxide in the presence of calf thymus DNA resulted in the formation of [14C]formamidine sulfinate ([14C]FASA), [14C]cyanomide, and [14C]urea and in covalent binding of radioactivity to the DNA. Incubation of V79 Chinese hamster cells with 10-20 mM TU for 18 hr but not for 3 hr, increased the frequency of micronuclei to a slight extent. In cells depleted of glutathione, which can prevent the oxidation of TU, micronucleus induction by TU was more pronounced and detectable both after 3 and 18 hr of incubation. Exposure of the cells to 1.25 to 10 mM FASA for 3-5 hr induced micronuclei, DNA repair synthesis, and gene mutations in the cells. Flavin-containing monooxygenase (FMO], an enzyme known to catalyze the S-oxygenation of TU in liver, could not be detected in the postmitochondrial supernatant (S-9) of the V79 cells. There is evidence, however, that TU can easily autoxidize to S-oxygenated products. Both FASA and TU caused a slight induction of DNA repair synthesis in cultured rat hepatocytes, but FASA was active at lower concentrations than TU. Cyanamide did not elicit repair. The finding that FASA, a product of both the nonenzymatic and the enzymatic S-oxygenation of TU, is genotoxic in cultured mammalian cells provides for the first time a hypothesis to explain the genotoxicity of TU.     

Covalent DNA adducts formed in mouse epidermis by benzo[g]chrysene

The metabolic activation in mouse skin of benzo[g]chrysene (B[g]C), a moderately carcinogenic polycyclic aromatic hydrocarbon (PAH) present in coal tar, was investigated. Male Parkes mice were treated topically with 0.5 micromol B[g]C and DNA was isolated from the treated areas of skin at various times after treatment and analysed by 32P-post-labelling. Seven major adduct spots were detected, at a maximum level of 6.55 fmol adducts/microg DNA. Mouse skin treated with the PAH benzo[c]phenanthrene (B[c]Ph) gave a total of 0.24 fmol adducts/microg DNA. B[g]C-DNA adducts persisted in skin for at least 3 weeks. Treatment of mice with 0.5 micromol of the optically pure putative proximate carcinogens, the (+)- and (-)-trans benzo[g]chrysene-11,12-dihydrodiols, led to the formation of adducts which comigrated on TLC and HPLC with those formed in B[g]C-treated mice, which suggested that the detected adducts were formed by the fjord region B[g]C-11,12-dihydrodiol-13,14-epoxides (B[g]CDEs). To test this, the four optically pure synthetic B[g]CDEs were reacted in vitro with DNA and the heteroco-polymers poly(dA x dT) and poly(dG x dC) and these samples 32P-postlabelled. Co-chromatography, on both TLC and HPLC, of in vitro and in vivo adducts indicated that B[g]C is activated in mouse skin through formation of the (-)-anti-(11R,12S,l3S,14R) and (+)-syn-(11S,12R,13S,14R) B[g]CDEs. (-)-anti-B[g]CDE formed five adducts with DNA, two of them with adenine and three with guanine bases. (+)-syn-B[g]CDE formed one adduct with each of these bases in DNA. The adenine adducts accounted for 64% of the total major adducts formed in B[g]C-treated mouse skin. The route of metabolic activation or B[g]C is similar to that reported for B[c]Ph, but the extent of activation to the fjord region diol-epoxides is significantly greater in the case of B[g]C, as demonstrated by the higher levels of adduct formation in vivo.     

Structure, conformations, and repair of DNA adducts from dibenzo[a, l]pyrene: 32P-postlabeling and fluorescence studies

The nature of stable DNA adducts derived from the very potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) in the presence of rat liver microsomes in vitro and in mouse skin in vivo has been studied using 32P-postlabeling and laser-based fluorescence techniques. Analysis of DB[a,l]P-DNA adducts via 32P-postlabeling has been obtained by comparison of the adduct patterns to those obtained from reactions of synthetic (+/-)-anti-, (+)-anti-, (-)-anti-, and (+/-)-syn-DB[a,l]P-11,12-diol 13,14-epoxide (DB[a,l]PDE) with single nucleotides and calf thymus DNA. anti-DB[a,l]PDE-dA adducts derived from the (-)-enantiomer are the major adducts formed in calf thymus DNA and in mouse skin DNA. The ratio of deoxyadenosine to deoxyguanosine modification is approximately 2:1 in mouse skin exposed to DB[a,l]P; activation by rat liver microsomes leads to a similar profile of adducts but with two additional spots. The conformations of DB[a,l]P adducts in native DNA, as well as the possibility of conformation-dependent repair, have been explored by low-temperature fluorescence spectroscopy. These studies have been performed using polynucleotides and calf thymus DNA reacted in vitro with DB[a,l]PDE and native DNA from mouse epidermis exposed to DB[a, l]P. The results show that adducts are heterogeneous, possess different structures, and adopt different conformations. External, external but base-stacked and intercalated adduct conformations are observed in calf thymus DNA and in mouse skin DNA samples. Differences in adduct repair rates are also revealed; namely, the analysis of mouse skin DNA samples obtained at 24 and 48 h after exposure to DB[a,l]P clearly shows that external adducts are repaired more efficiently than intercalated adducts. These results, taken together with those for B[a]P-DNA adducts [Suh et al. (1995) Carcinogenesis 16, 2561-2569], indicate that the repair of DNA damage resulting from PAH diol epoxides is conformation-dependent.     

Human melanocytes and keratinocytes exposed to UVB or UVA in vivo show comparable levels of thymine dimers

Epidemiology shows a relationship between solar exposure and all types of skin cancer. Understanding the mechanisms of skin cancer requires knowledge of the photomolecular events that occur within the relevant epidermal cell types in vivo. Studies to date have focused on UVR-induced DNA lesions in keratinocytes, the majority epidermal cell population which gives rise to most skin cancers. Malignant melanoma, arising from melanocytes (5%-10% of epidermal cells), accounts for most skin cancer deaths. We report on new techniques to detect DNA photolesions in human epidermal melanocytes in situ. Previously nonexposed buttock skin of volunteers of skin types I/II was exposed to clinically relevant doses of narrow bandwidth UVB (300 nm) and UVA (320 nm, 340 nm, 360 nm) radiation. Biopsies were taken immediately afterwards and processed for routine histology. Microscope sections were prepared and double-stained with fluorescent-tagged monoclonal antibodies for thymine dimers and melanocytes. UVR dose-response curves for dimer levels within melanocyte nuclei were determined by image analysis and compared with dimer levels in adjacent basal cell keratinocytes. Our data show that UVB and UVA readily induce thymine dimers in melanocytes at levels that are comparable with those found in adjacent keratinocytes. This new technique will enable melanocyte specific studies, such as DNA repair kinetics, to be done in vivo.     

Risk assessment of dithiocarbamate accelerator residues in latex-based medical devices: genotoxicity considerations

The Medical Devices Agency (MDA) has investigated potential human health hazards arising from the presence of dithiocarbamate vulcanization accelerators in latex products (mainly gloves). After collection of manufacturer's data on usage and residues of these accelerators, an independent investigation of solvent extractable residues and dithiocarbamate migration into aqueous simulants was commissioned, to complement equivalent "in-house" test data from two major manufacturers. The presence of extractable accelerator residues in commercial products was confirmed. Potential human health hazards associated with dithiocarbamates include genotoxicity and possible carcinogenicity: a review of published data was conducted to evaluate the evidence for this, with particular reference to three zinc dithiocarbamates with significant commercial usage (ZDMC, ZDEC and ZDBC: see Fig. 1). Data gaps were identified, and mutagenicity studies commissioned to fill these. These studies comprised tests both in vitro (bacterial and L5178Y cell gene mutation, cultured lymphocyte chromosome aberration) and in vivo (mouse bone marrow micronucleus, rat liver UDS). It is concluded that ZDMC must be considered a genotoxin (and thus a probable carcinogen): residues of this substance in latex medical devices should be minimized. ZDEC proved genotoxic in vitro but was not clearly genotoxic in vivo, and may have activity intermediate between that of ZDMC and that of ZDBC, which showed at most weak activity in a single in vitro (chromosome aberration) test. It is proposed that the use of ZDBC as a vulcanization accelerator in the manufacture of latex gloves, rather than ZDEC, ZDMC or their precursors, would reduce or remove the health concerns arising from accelerator residues.     

The usefulness of three biallelic restriction fragment length polymorphisms versus a polymorphic dinucleotide tandem repeat polymorphism at the low-density-lipoprotein receptor gene locus for diagnosis of familial hypercholesterolemia

The DNA adduct 8-hydroxy-2'-deoxyguanosine (8-OHdG) has been widely used as a biomarker for oxidative stress. Bulky DNA adducts, which are detectable by the 32P-postlabelling method, provide evidence for exposure to and metabolic activation of large, mainly apolar compounds, e.g. polycyclic aromatic hydrocarbons. We determined both types of adducts in placental tissues of 30 term pregnancies and related the adduct levels to the exposure to tobacco smoke and the plasma antioxidant status. Urine and plasma continine concentrations were used to select 10 nonsmokers, 9 nonsmokers exposed to environmental tobacco smoke (ETS) and 11 smoking women. Placental levels of 8-OHdG were 0.84 +/- 0.11, 0.90 +/- 0.21 and 0.83 +/- 0.20/10(5) deoxyguanosine bases (dG) for nonsmokers, nonsmokers exposed to ETS and smokers, respectively. The differences between the groups were not significant. Smoking women had significantly lower plasma vitamin C and beta-carotene concentrations than nonsmoking women or nonsmoking women exposed to environmental tobacco smoke. The 8-OHdG adduct level in placental DNA was inversely correlated with the plasma vitamin E concentration (r = -0.47, P < 0.05). There was no association between placental 8-OHdG adducts and vitamin A, C and beta-carotene in plasma. In total, 15 different adducts could be identified in the 30 placenta samples by the 32P-postlabelling method. There was a strong inter-individual variation in both the number of adducts and adduct intensities. No smoking-related or vitamin-related effects on adduct patterns or intensities were found. Our findings suggests that, within the limits of the methods used, tobacco smoke exposure during pregnancy does not lead to a measurable increase in placental DNA adduct levels and that vitamin E appears to have a protective effect on placental 8-OHdG formation.     

Use of metabolically competent human hepatoma cells for the detection of mutagens and antimutagens

The human hepatoma line (Hep G2) has retained the activities of various phase I and phase II enzymes which play a crucial role in the activation/detoxification of genotoxic procarcinogens and reflect the metabolism of such compounds in vivo better than experimental models with metabolically incompetent cells and exogenous activation mixtures. In the last years, methodologies have been developed which enable the detection of genotoxic effects in Hep G2 cells. Appropriate endpoints are the induction of 6-TGr mutants, of micronuclei and of comets (single cell gel electrophoresis assay). It has been demonstrated that various classes of environmental carcinogens such as nitrosamines, aflatoxins, aromatic and heterocyclic amines and polycyclic aromatic hydrocarbons can be detected in genotoxicity assays with Hep G2 cells. Furthermore, it has been shown that these assays can distinguish between structurally related carcinogens and non-carcinogens, and positive results have been obtained with rodent carcinogens (such as safrole and hexamethylphosphoramide) which give false negative results in conventional in vitro assays with rat liver homogenates. Hep G2 cells have also been used in antimutagenicity studies and can identify mechanisms not detected in conventional in vitro systems such as induction of detoxifying enzymes, inactivation of endogenously formed DNA-reactive metabolites and intracellular inhibition of activating enzymes.     

Endogenous skin fluorescence includes bands that may serve as quantitative markers of aging and photoaging

Aging and photoaging cause distinct changes in skin cells and extracellular matrix. Changes in hairless mouse skin as a function of age and chronic UVB exposure were investigated by fluorescence excitation spectroscopy. Fluorescence excitation spectra were measured in vivo, on heat-separated epidermis and dermis, and on extracts of mouse skin to characterize the absorption spectra of the emitting chromophores. Fluorescence excitation spectra obtained in vivo on 6 wk old mouse skin had maxima at 295, 340, and 360 nm; the 295 nm band was the dominant band. Using heat separated tissue, the 295 nm band predominantly originated in the epidermis and the bands at 340 and 360 nm originated in the dermis. The 295 nm band was assigned to tryptophan fluorescence, the 340 nm band to pepsin digestable collagen cross-links fluorescence and the 360 nm band to collagenase digestable collagen cross-links fluorescence. Fluorescence excitation maxima remained unchanged in chronologically aged mice (34-38 wk old), whereas the 295 nm band decreased in intensity with age and the 340 nm band increased in intensity with age. In contrast, fluorescence excitation spectra of chronically UVB exposed mice showed a large increase in the 295 nm band compared with age-matched controls and the bands at 340 and 350 nm were no longer distinct. Two new bands appeared in the chronically exposed mice at 270 nm and at 305 nm. These reproducible changes in skin autofluorescence suggest that aging causes predictable alterations in both epidermal and dermal fluorescence, whereas chronic UV exposure induces the appearance of new fluorphores.     

Factors affecting the genotoxic potency ranking of natural anthraquinones in mammalian cell culture systems

We had reported that the plant-derived 1,8-dihydroxyanthraquinone derivatives, emodin and danthron, were clearly genotoxic in mouse lymphoma L5178Y cells, whereas chrysophanol was only weakly genotoxic and physcion not at all. Danthron was more potent than emodin. Furthermore, we had found that these compounds bound non-covalently to DNA and inhibited topoisomerase II activity. Interestingly, in these systems emodin was more potent than danthron. This inversion of the ranking prompted us to investigate the underlying mechanism. Since emodin shows a high serum-protein binding affinity, horse serum used as a media-supplement in the mouse lymphoma genotoxicity assays was analyzed for a potential selective scavenging of emodin. Non-covalent DNA-binding in mouse lymphoma L5178Y cells was investigated in the absence or presence of serum. In the presence of 10% serum, the DNA-binding potency of emodin was markedly reduced and was lower than that of danthron. We also applied mutation assays with mouse lymphoma cells and AS52 cells and varied the serum concentration used. In the absence of serum emodin showed slightly higher mutagenicity in AS52 cells than danthron. At reduced serum concentration (0.5%) emodin was strongly cytotoxic to the mouse lymphoma cells. For chrysophanol and physcion, a considerable reduction of the non-covalent DNA-binding potency in intact cells was found when compared to danthron, in concordance with their lower genotoxic potency. Overall, these data support the understanding that the genotoxicity of anthraquinones is, at least in part, mediated by non-covalent DNA-binding.