Melanin offers protection against induction of cyclobutane pyrimidine dimers and 6-4 photoproducts by UVB in cultured human melanocytes.

The goal of this investigation was to correlate the melanin content in human pigmentary cells with the generation of UVB-induced photoproducts and to examine the relationship between the melanin content and the removal of the photoproducts. Cultured melanocytes from light-skinned individuals synthesized less melanin and produced more cyclobutane pyrimidine dimers and 6-4 photoproducts upon UVB exposure than did melanocytes from black skin. Tyrosine-stimulated melanogenesis provided protection against DNA damage in both cell types. In another set of pigmented cell lines a ratio between eumelanin and pheomelanin was determined. The assessment of association between DNA damage induction and the quantity and quality of melanin revealed that eumelanin concentration correlated better with DNA protection than pheomelanin. Skin type-I and skin type-VI melanocytes, congenital nevus (CN)-derived cells and skin type-II melanocytes from a multiple-melanoma patient were grown in media with low or high L-tyrosine concentration. The cells were irradiated with 200 J/m2 UVB, and the levels of the photoproducts were determined immediately and after 6 and 24 h. Once again the induction of the photoproducts was mitigated by increased melanogenesis, and it was inversely correlated with the skin type. No significant differences were found for the removal of photoproducts in the cultures of skin types I and VI and CN cells. No indications of a delay in the removal of photoproducts in the melanocytes from the multiple-melanoma patient were found either.

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.

Cell proliferation and DNA adducts in hamster tracheal epithelium exposed to benzo[a]pyrene in organ culture.

The effect of benzo[a]pyrene (B[a]P) on cell proliferation in cultured hamster tracheal epithelium was studied in relation to the formation of B[a]P-DNA adducts. To this end, tracheae were isolated from Syrian golden hamsters, cut into rings and cultured in Ham's F12 medium. Then, the tracheal rings were exposed to 2 or 20 mumB[a]P, either continuously for 7 days or for 2 days followed by a 5-day recovery period without B[a]P. At intervals rings were sampled for determination of cell proliferation (by means of the labelling index). In addition, B[a]P-DNA adduct levels were determined in the continuous exposure experiment by means of in situ detection by immunofluorescence microscopy. After 2 days of exposure to 2 or 20 mum B[a]P, there was a significant increase in B[a]P-DNA adduct level. A further, linear increase in B[a]P-DNA adduct level, however, was only observed after continuous exposure to 20 mum B[a]P, whereas the adduct level in the 2 mum continuous exposure group remained virtually the same. In unexposed tracheal epithelium an initial peak of cell proliferation was observed. This initial proliferation was significantly lower in the exposed samples. Only continuous exposure to 20 mum B[a]P steadily decreased the labelling index from day 2 to 7. It is concluded that the increase in B[a]P-DNA adduct level is correlated with the reduction of cell proliferation in hamster tracheal epithelium exposed to B[a]P in Ham's F12 medium.

Molecular dosimetry of DNA damage induced by polycyclic aromatic hydrocarbons; relevance for exposure monitoring and risk assessment.

Polycyclic aromatic hydrocarbons (PAH) form a large group of organic chemicals that are widely distributed in our environment as pollutants of air, water and soil. Several PAH are carcinogenic in rodents, while exposure to these compounds has been associated with various types of human cancer. Upon entering the body, PAH may be converted into reactive electrophilic species, which can give rise to the formation of DNA adducts. DNA adduct formation is considered to be the initial event in chemical carcinogenesis. In this paper, two methods are illustrated that are widely used to determine PAH-DNA adduct formation, namely 32P-postlabelling, and immunochemical analysis with specific antibodies. The applications of the 32P-postlabelling assay comprise the following: A study of interspecies differences in PAH bioactivation in vitro, with microsomal preparations isolated from liver tissue of various rodent species and of human origin; the results indicate that there are considerable qualitative differences between the adduct patterns obtained, which is relevant with respect to extrapolation from animal to man. The analysis of DNA adduct formation in fish retrieved from marine environments polluted to various extents with PAH; results of these studies show a correlation between liver-DNA adduct levels in these fish and the degree of PAH contamination in the aquatic environment. Biomonitoring of PAH exposure through analysis of adducts in blood cells obtained from heavy and light smokers; the data show a fair correlation between PAH-DNA adduct levels in white blood cells and cotinine content in blood plasma, the latter being used as a marker for exposure to cigarette smoke.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of DNA adducts in basal and non-basal cells of the hamster trachea exposed to benzo(a)pyrene in organ culture.

DNA adducts were quantified in hamster tracheas exposed to benzo(a)pyrene (BP) in organ culture, in basal as well as in non-basal cells, by in situ detection with an adduct-specific rabbit antiserum (W2/01) and with a mouse monoclonal antibody against human cytokeratins 5 and 8 (RCK102) to identify hamster trachea basal cells. Recognition by W2/01 of the adduct of (+)-anti-7,8-dihydroxy-9,10-epoxide of BP (BP-diolepoxide; BPDE) to deoxyguanosine (dG) was checked on human white blood cells (WBCs) exposed to BP together with 3-methylcholanthrene (3MC)-induced rat-liver microsomes. By comparison with the adduct levels determined by 32P post-labeling, a lower detection limit of about 1 adduct per 10(6) nucleotides could be deduced. Next, tracheal rings were exposed to BP (40 microM) in organ culture for 2 days, then washed and cultured without BP for another 3 days. At different time points epithelial cells were isolated and cytospin preparations made. Staining of BP DNA adducts combined with that of cytokeratin (both visualized with fluorescence) allowed detection of adducts in both basal and non-basal cells in the same preparation. BP DNA adduct formation in basal and non-basal cells after 2 days of exposure to BP was not different. However, on removal of BP the adducts disappeared significantly faster from basal cells than from non-basal cells. The combination of the two antibodies mentioned above thus allows selective determination of BP DNA adduct levels in different cell types. This could be of importance with regard to the involvement of specific cell types in the process of tumor initiation.

Class-specific immunoadsorption purification for polycyclic aromatic hydrocarbon-DNA adducts.

An immunoenrichment procedure has been developed for applications in the detection and identification of a broad range of polycyclic aromatic hydrocarbon (PAH)-DNA adducts at very low abundance. The procedures are based on a monoclonal antibody raised to r-7,trans-8,trans-9-trihydroxy-cis-10-(N2-deoxyguanosyl-5'-phospha te)- 7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-N2-dG) which has been tested for cross-reactivity towards DNA and proteins (bovine serum albumin, chicken gamma globulin and human globin) covalently modified with a range of PAH diol-epoxides. The antibody recognised DNA adducted with the diol-epoxides of benzo[a]pyrene, benz[a]anthracene, chrysene, dibenz[a,h]anthracene or picene. The antibody also cross-reacted with the 7,8,9,10-tetraol derived from benzo[a]pyrene and the 1,2,3,4-tetraols of benz[a]anthracene and chrysene. The degree of cross-reactivity was greatest for PAH adducts with structural features similar to anti-BPDE-N2-dG proximate to the base attachment. The antibody also recognised a range of PAHs adducted to human globin; these included adducts of benzo[a]pyrene, benz[a]anthracene and chrysene diol-epoxides. This wide range of recognition provides good evidence for the class-specific recognition of PAH adducts by the antibody. When immobilized on Sepharose 4B and used in the immunoadsorption purification of adducted nucleotides, the antibody selectively enriched adducts of benzo[a]pyrene, benz[a]anthracene and chrysene from normal nucleotides. Quantitative measurements with [14C]benzo[a]pyrene-DNA adducts showed that the immobilized antibody was able to enrich benzo[a]pyrene adducts from a DNA hydrolysate containing adducts at a level of 1 adduct/10(10) normal nucleotides. In addition, this immunoadsorption technique was effective in enriching a mixture of DNA adducts formed in the skin of CF1 mice treated cutaneously with a mixture of [3H]benzo[a]pyrene and [3H]chrysene. Class-specific immunoenrichment procedures for DNA adducts are important in assisting the identification of genotoxic components in complex mixtures. The performance characteristics of this immobilized antibody suggest that it may be suitable for application in the detection, identification and monitoring of human exposures to low levels of PAHs.

DNA adducts in hamster and rat tracheas exposed to benzo(a)pyrene in vitro.

Syrian golden hamsters are much more susceptible than Wistar rats to the induction of tracheal tumors by benzo(a)pyrene (BP). In order to investigate whether this difference is reflected in the pattern of DNA-adduct induction and removal, tracheas from either species were isolated and exposed to BP (5 micrograms/ml) in organ culture. At various time-points BP-DNA adducts in the epithelial cells were quantified by 32P-postlabeling; unscheduled DNA synthesis (UDS) was determined by [3H]thymidine incorporation. In an induction-repair experiment tracheas were exposed to BP for 2 days, and cultured for another 4 days without BP. After 2 days of exposure total BP-DNA adduct levels were 10 times higher in hamster compared to rat tracheas. In hamster tracheas one major adduct was formed (95%), vs. the adduct between (+)-anti-BP-diolepoxide and deoxyguanosine (BPDE-N2dG). In rat tracheas BPDE-N2dG comprised about 60% of the total adduct level. During exposure to BP the adduct level in hamster trachea increased to 36 +/- 19 adducts/10(6) nucleotides (add/10(6) n) on day 2. Two days after removal of BP the BP-DNA adduct level had decreased to 60% of that on day 2; there was no further decrease in the BP-DNA adduct level. UDS increased during exposure to BP and decreased after removal of BP. In rats, removal of BP did not lead to a decrease in the BP-DNA adduct level, which agreed with the observed absence of UDS. In a second experiment tracheas were exposed to BP continuously for 15 days. In hamster tracheas the total BP-DNA adduct level increased from 11 +/- 0.7 add/10(6) n after 1 day of exposure to 105 +/- 2 add/10(6) n after 15 days; also UDS increased with increasing exposure until day 11. In rat tracheas no progressive increase in the BP-DNA adduct level was seen. It was concluded that the difference in trachea tumor susceptibility between hamsters and rats exposed to BP correlates with the difference between the 2 species in BP-DNA adduct kinetics in the trachea epithelial cells.

Detection of benzo[a]pyrene-DNA adducts in cultured cells treated with benzo[a]pyrene diol-epoxide by quantitative immunofluorescence microscopy and 32P-postlabelling; immunofluorescence analysis of benzo[a]pyrene-DNA adducts in bronchial cells from smoking individuals.

Monoclonal antibodies were raised against the reaction product of benzo[a]pyrene diol-epoxide (BPDE) and deoxyguanosine-5'-monophosphate. The antibodies were used for detection of DNA adducts in situ in BPDE-treated cultured human fibroblasts by immunofluorescence microscopy. Analogue-digital conversion of the fluorescence signal and further image processing allowed measurement of the immunospecific fluorescence in the nuclei of these cells. The results are compared with the adduct levels measured in isolated DNA by 32P-postlabelling. Preliminary results are shown of the application of the immunofluorescence method to the analysis of DNA adducts in bronchial cells obtained from smoking individuals.