Building confidence in skin sensitisation potency assessment using new approach methodologies: report of the 3rd EPAA Partners Forum, Brussels, 28th October 2019.

Skin sensitising substances that induce contact allergy and consequently risk elicitation of allergic contact dermatitis (ACD) remain an important focus regarding the replacement of animal experimentation. Current in vivo methods, notably the local lymph node assay (LLNA) refined and reduced animal usage and led to a marked improvement in hazard identification, characterisation and risk assessment. Since validation, regulatory confidence in the LLNA approach has evolved until it became the first choice assay in most regulated sectors. Currently, hazard identification using the LLNA is being actively replaced by a toolbox of non-animal approaches. However, there remains a need to increase confidence in the use of new approach methodologies (NAMs) as replacements for LLNA sensitiser potency estimation. The EPAA Partners Forum exchanged the current state of knowledge on use of NAMs in various industry sectors and regulatory environments. They then debated current challenges in this area and noted several ongoing needs. These included a requirement for reference standards for potency, better characterisation of applicability domains/technical limitations of NAMs, development of a framework for weight of evidence assessments, and an increased confidence in the characterisation of non-sensitisers. Finally, exploration of an industry/regulator cross-sector user-forum on skin sensitisation was recommended.

The isolated chicken eye test as a suitable in vitro method for determining the eye irritation potential of household cleaning products.

Eye irritation is an important endpoint in the safety evaluation of consumer products and their ingredients. Several in vitro methods have been developed and are used by different industry sectors to assess eye irritation. One such in vitro method in use for some time already is the isolated chicken eye test (ICE). This investigation focuses on assessing the ICE as a method to determine the eye irritation potential of household cleaning products, both for product safety assurance prior to marketing and for classification and labeling decisions. The ICE involves a single application of test substances onto the cornea of isolated chicken eyes. Endpoints are corneal swelling, corneal opacity and fluorescein retention. The ICE results were compared to historic LVET data in this study due to availability of such in vivo data and the ability to correlate LVET to human experience data on the outcome of accidental exposures to household cleaning products in general. The results of this study indicate that the ICE test is a useful in vitro method for evaluating the eye irritation/corrosion potential and establishing classification and labeling for household cleaning products. For new product formulations, it is best used as part of a weight-of-evidence approach and benchmarked against data from comparable formulations with known eye irritation/corrosion profiles and market experience.

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.

Vitamin A and beta-carotene influence the level of benzo[a]pyrene-induced DNA adducts and DNA-repair activities in hamster tracheal epithelium in organ culture.

Although most studies concerning the effect of vitamin A and beta-carotene on chemical carcinogenesis are focused on tumour promotion and progression, these compounds may affect initiation as well. In this study the influence of vitamin A and beta-carotene on unscheduled DNA synthesis (UDS) was investigated in hamster tracheal epithelium in organ culture exposed to benzo[a]pyrene (B[a]P). DNA-repair activities were compared with the level of B[a]P-DNA adducts as measured both by 32P-postlabeling and by immunocytochemical detection. In hamster tracheal epithelial cells, both vitamin A and beta-carotene significantly increased B[a]P-induced UDS, with 40% and 45%, respectively. At the same time, vitamin A and beta-carotene decreased the level of B[a]P-DNA adducts in these cells with 18% and 40%, respectively as measured by 32P-postlabeling and with 12% and 35%, respectively as measured by immunocytochemistry. The effect of vitamin A on B[a]P-induced UDS and DNA-adduct levels in hamster tracheal epithelium appeared to depend on the dose of B[a]P vis-à-vis the concentration of vitamin A. The results of the present study show that both vitamin A and beta-carotene cause a decrease in B[a]P-DNA adduct levels by enhancing DNA-repair activities. Because the formation of B[a]P-DNA adducts is considered to be an early step in respiratory tract carcinogenesis, it is suggested that enhancement of DNA-repair activities by vitamin A and the subsequent removal of DNA adducts may be one of the mechanisms involved in vitamin A-mediated protection against cancer.

Skin sensitization testing in potency and risk assessment.

The purpose of this article is to review, and make recommendations for, the use of relevant skin sensitization test methods, for the purposes of determination of relative potency and the threshold dose necessary for the induction of skin sensitization, and for risk assessment. In addressing the first area, the utility of three guinea pig tests (the guinea pig maximization test, the occluded patch test, and the open epicutaneous test) of the local lymph node assay (LLNA) and of human volunteer testing for the assessment of relative potency and identification of thresholds for sensitization were considered. The following conclusions were drawn. (1) Although attempts have been made to modify the guinea pig maximization test for the purposes of deriving dose-response relationships, this method is usually unsuitable for determination of relative sensitizing potency. (2) Guinea pig methods that do not require the use of adjuvant and which employ a relevant route of exposure (the occluded patch test and the open epicutaneous test) are more appropriate for the assessment of relative skin-sensitizing potency. (3) The LLNA is suitable for the determination of relative skin sensitizing potency, and the adaptation of this method for derivation of comparative criteria such as EC3 values (the estimated concentration of test chemical required to induce a stimulation index of 3 in the LLNA) provides an effective and quantitative basis for such measurements. (4) For all the methods identified above, potency is assessed relative to other chemical allergens of known skin sensitizing potential. The estimation of likely threshold concentrations is dependent upon the availability of suitable benchmark chemicals of known potency for human sensitization. (5) Human testing (and specifically, the Human Repeat Insult Patch Test) can provide information of value in confirming the absence of skin sensitizing activity of formulations and products under specific conditions of use and exposure. Based on the above, the following recommendations are made. (1) If results are already available from suitable guinea pig tests, then judicious interpretation of the data may provide information of value in assessing relative skin sensitizing potency. This option should be explored before other analyses are conducted. (2) The LLNA is the recommended method for new assessments of relative potency, and/or for the investigation of the influence of vehicle or formulation on skin sensitizing potency. (3) Whenever available, human skin sensitization data should be incorporated into an assessment of relative potency. With respect to risk assessment, the conclusion drawn is that all the available data on skin-sensitizing activity in animals and man should be integrated into the risk-assessment process. Appropriate interpretation of existing data from suitable guinea pig studies can provide valuable information with respect to potency, as the first step in the development of a risk assessment. However, for de novo investigations, the LLNA is the method favored for providing quantitative estimations of skin-sensitizing potency that are best suited to the risk assessment process. Finally, human testing is of value in the risk assessment process, but is performed only for the purposes of confirming product safety.

32P-postlabelling analysis of DNA adducts in white blood cells of humans exposed to residential wood combustion particulate matter.

Residential wood combustion (RWC) in open fireplaces poses a possible health risk because of the emission into the indoor air of mutagenic and carcinogenic compounds. In the present report it was investigated whether this emission leads to enhanced levels of DNA adducts in white blood cells (WBC) of exposed subjects. Under conditions that most likely reflect the Dutch pattern of use of open fireplaces, RWC increased both indoor air mutagenicity and levels of benzo(a)pyrene (B(a)P) and pyrene. The indirect mutagenicity showed a stronger increase than the direct mutagenicity. The increase in indirect mutagenicity was not directly correlated with the increase in the levels of B(a)P and pyrene. 32P-postlabelling analysis of DNA adducts following nuclease P1 enrichment or butanol extraction revealed low adduct levels. No combustion-related increase in the amount of adducts was observed. Possible explanations for the lack of correlation between air monitoring data and WBC DNA adduct levels are discussed.

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.

The use of benzo[a]pyrene diolepoxide-modified DNA standards for adduct quantification in 32P-postlabelling to assess exposure to polycyclic aromatic hydrocarbons: application in a biomonitoring study.

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.

Influence of growth factors and medium composition on benzo[a]pyrene- and vitamin A-induced cell proliferation and differentiation in hamster tracheal epithelium in organ culture.

Tracheal organ cultures and isolated tracheal epithelial cells are frequently used to study effects of carcinogens and retinoids on both proliferation and differentiation of respiratory tract epithelial cells. For each of these in vitro models, optimal culture conditions have been established, varying in type of culture medium and composition of growth factor and hormone supplementation, which by themselves may influence cellular proliferation and differentiation. In this study, we investigated the influence of medium composition and growth factor supplementation on the effect of benzo[a]pyrene (B[a]P) and vitamin A on cellular proliferation and differentiation in hamster tracheal epithelium in organ culture. In tracheae cultured in Ham's F12 medium, cell proliferation was decreased by B[a]P relative to untreated controls, whereas vitamin A in combination with B[a]P increased cell proliferation compared with that in tracheae treated with B[a]P alone. The effects in tracheae cultured in CMRL-1066 medium were just the opposite: B[a]P increased cell proliferation and vitamin A decreased B[a]P-induced proliferation. To explain this difference in cell proliferation, the effects of various growth factors (epidermal growth factor and transferrin) and medium components (nucleotides, NAD(+)/NADP and CaCl(2).2H(2)O) on B[a]P and vitamin A-induced cell proliferation were investigated. The main factor responsible for the different effects on cell proliferation appeared to be the concentration of Ca(2+) in the culture medium; addition of CaCl(2).2H(2)O to Ham's F12 medium resulted in effects of B[a]P and vitamin A on cell proliferation comparable with those observed in tracheae cultured in CMRL-1066 medium. These results clearly show that the composition of the culture medium, and particularly the concentration of Ca(2+), strongly influences the effect of vitamin A and B[a]P on cell proliferation in hamster tracheal epithelium in organ culture.

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.

Eye irritation responses in rabbit and man after single applications of equal volumes of undiluted model liquid detergent products.

A criticism of the use of the rabbit low-volume eye test to determine eye irritation hazard for man is the lack of comparative data in man and rabbit with undiluted products. To address this, such a study has been performed in man and rabbit using undiluted model liquid detergents. The hypothesis tested was that if, under identical test conditions, the effects in the rabbit were the same or greater than the response in man, then it is valid to use the low-volume eye test to assess eye irritation hazard for man. The studies were carried out using 29 human volunteers and 12 rabbits. The effects in the rabbit were unequivocally greater than the effects observed in man, but clearly less than the expected response from these types of product in the Draize test. The results from this study confirm the sensitivity of the rabbit as a test species, and support the use of the low-volume eye test to assess eye irritation hazard for man. Any in vitro/ex vivo alternative to assess eye irritation should be developed against the rabbit low-volume eye test or human data where available.

Skin sensitisation testing--new perspectives and recommendations.

Various methodological aspects of skin sensitisation testing have been explored, particularly in the context of animal welfare considerations and reliability and sensitivity of test methods. Recommendations are made for the conduct of current and proposed OECD skin sensitisation tests with respect to appropriate test configurations for the purposes of hazard identification and labelling, and the requirement for positive controls. Specifically, the following aspects of guinea pig sensitisation test methods have been addressed: (1) the number of test and control animals required; (2) the option of using joint positive controls between independent laboratories; (3) the choice of positive control chemicals; (4) the optimal conduct and interpretation of rechallenge; and (5) the requirement for pretreatment with sodium lauryl sulfate. In addition, the use of the murine local lymph node assay (LLNA) has been considered. A number of conclusions have been drawn and recommendations made as follows: In many instances, particularly with the conduct of the guinea pig maximisation test, it is acceptable to halve the number of test and control animals used. An optional scheme for the conduct of joint positive control studies within a co-ordinated group of laboratories is appropriate. Only one positive control chemical (alpha-hexyl cinnamic aldehyde) is necessary for the routine assessment of assay sensitivity. The proper conduct and interpretation of rechallenge can provide valuable information and confirmation of results in guinea pig sensitisation tests. Sodium lauryl sulfate should no longer be used as a pretreatment in the guinea pig maximisation test. The LLNA is a viable and complete alternative to traditional guinea pig test methods for the purposes of skin sensitisation hazard identification. These recommendations provide the opportunity for both animal welfare benefits and improved hazard identification.

Human volunteer studies; a consumer products company view.

The term 'contact dermatitis' refers to a range of adverse effects whose causation is quite varied. Manufacturers of consumer products have an important responsibility to minimise the extent to which their products cause such skin reactions. In meeting this responsibility, use may be made of humans, e.g. in studies related to skin irritation, to try to ensure the highest possible safety standards are achieved. The purpose of this short review paper is to outline the principles that must be followed before initiating studies with human volunteers. In addition, these principles are considered in the context of European legislation on chemicals and preparations.

Designation of substances as skin sensitizing chemicals: a commentary.

During the last 10 years there have been several attempts to define criteria for the integration of experimental and clinical data into schemes that can be used for the designation of chemicals as skin sensitizers (and in some instances respiratory allergens). The last such proposal was made recently in this journal by Schnuch and colleagues (Hum Exp Toxicol 2002; 2: 439-44) who invited critical discussion and debate of the area. In this present article we have sought to build upon and refine further those previous recommendations and suggest here a modified scheme for the classification of chemicals as confirmed or probable skin sensitizers. This new scheme we believe provides a realistic framework within which informed decisions can be reached about likely skin sensitizing activity based upon judicious consideration of clinical and experimental information.

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)

Relation between benzo[a]pyrene-DNA adducts, cell proliferation and p53 expression in tracheal epithelium of hamsters fed a high beta-carotene diet.

Vitamin A and beta-carotene protect against respiratory tract cancer by inhibiting the formation of DNA damage and controlling cellular proliferation and differentiation. Recently, it has been shown that the p53 tumor-suppressor gene plays a crucial role in the etiology of respiratory tract cancer. In the present study, we investigated the relationship between benzo[a]pyrene (B[a]P)-DNA adducts, cell proliferation and p53 expression and the possible effect of beta-carotene on such a relationship in tracheal epithelium of hamsters given intratracheal instillations of B[a]P-Fe2O3 particles suspended in saline. DNA-adducts were quantified by the 32P-postlabeling assay, cell proliferation was quantified by immunocytochemical detection of incorporated BrdU during S-phase, and p53 protein was detected by immunohistochemistry with an antibody that recognized both the wild-type and the mutated protein (BioGenex, Clone BP53-12-1). A clear relationship appeared to exist between the extent of B[a]P-DNA adduct formation, the induction of cell proliferation and the expression of p53 protein in hamster tracheal epithelium. These results suggest that B[a]P induces cell proliferation in hamster tracheal epithelial cells most likely by the induction of mutations in the p53 gene. Furthermore, beta-carotene was not found to influence the formation of B[a]P-DNA adducts, which is probably due to the high B[a]P dose. Moreover, beta-carotene did not statistically significantly affect cell proliferation and p53-protein expression in hamster tracheal epithelial cells.

Comparative 32P-postlabeling analysis of benzo[a]pyrene--DNA adducts formed in vitro upon activation of benzo[a]pyrene by human, rabbit and rodent liver microsomes.

An interspecies comparison was made of the DNA-adducts formed in vitro upon incubation of rat liver DNA (RL-DNA) with benzo[a]pyrene (BP) in the presence of liver microsomes. Incubations were carried out with RL-DNA, BP (100 microM) and liver microsomes from hamsters, mice, rabbits, rats, 3-methylcholanthrene (3MC) pretreated rats and from humans. To analyse the adduct profiles, the 32P-postlabeling technique with the nuclease P1-enhancement procedure was used. The total amount of adduct formed varied greatly with the species; also the number of adduct spots detected was different, ranging from 1 to 5. In all incubations the BP-N2-deoxyguanosine adduct was formed. Relative to the total adduct level, the level of this adduct varied from 26% with rat, 54% with hamster, 56% with 3MC-pretreated rat, 58% with mouse and 75% with rabbit, to 100% with human liver microsomes. In human liver microsomes both the total amount of cytochrome P-450 per mg microsomal protein and the ethoxyresorufin O-deethylation (EROD) activity were low compared to that in animal liver microsomes. In microsomes from 3MC-pretreated rats the EROD activity was strongly induced. There was no correlation between EROD activity in non-induced microsomes and total adduct level. To compare BP-DNA adduct formation in human white blood cells (WBC) with that in RL-DNA, WBC were incubated with BP and 3MC-pretreated rat microsomes. The adduct profile in WBC-DNA differed from that observed after incubation of RL-DNA: the BP-N2-deoxyguanosine adduct in WBC-DNA accounted for 97% of the total adduct level. It is concluded that the 32P-postlabeling method is a suitable technique to investigate both qualitative and quantitative differences in BP-DNA adduct formation between species. Furthermore, the incubation of microsomes from the liver (or other sources) with a genotoxic agent and isolated DNA or cells can be a useful approach to study the formation and stability of reactive intermediates that are able to bind to DNA, also with respect to differences between species or tissue.

Formation and repair of benzo[a]pyrene-DNA adducts in cultured hamster tracheal epithelium determined by 32P-postlabeling analysis and unscheduled DNA synthesis.

Hamster tracheal organ cultures were used to investigate the relationship between DNA adduct formation measured directly by the 32P-postlabeling assay, and the DNA damage measured indirectly by the unscheduled DNA synthesis (UDS) assay. Hamster tracheas were treated with three concentrations of benzo[a]pyrene (B[a]P) for 2 days. Postlabeling and UDS assays were also carried out a few days after removal of the B[a]P. Furthermore, the types of B[a]P-DNA adducts formed in the in vitro organ culture were qualitatively compared with those formed in vivo after intratracheal intubation of B[a]P attached to Fe2O3 particles. In vivo only one adduct was detected by 32P-postlabeling. This adduct cochromatographed with the trans-addition produce of dG and (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). In vitro, a clear B[a]P-DNA adduct pattern was also found with the 32P-postlabeling assay. Four different adducts were found. The main adduct spot migrated to the same position on the thin-layer chromatogram as the in vivo adduct. B[a]P-DNA adduct formation was both time- and dose-dependent. During the first day after removal of B[a]P the adduct levels still increased, thereafter they decreased at all B[a]P concentrations. A time- and dose-dependent increase in UDS was observed in the tracheal epithelial cells treated with B[a]P in vitro. After removal of the B[a]P, UDS decreased immediately, in contrast to the formation of DNA adducts. The results of the present study show that B[a]P induces time- and dose-dependently both DNA adducts and UDS in hamster tracheal organ culture. Moreover, the main DNA adduct formed in vitro, dG-(+)-anti-BPDE, was the same as that found in vivo.

Improvements in the 32P-postlabelling procedure to quantify bulky aromatic DNA adducts.

This contribution describes methodological modifications and improvements that may contribute to inter-assay reproducibility and more accurate adduct quantification for 32P-postlabelling. Firstly, an anion-exchange chromatography procedure was developed to determine the amount of DNA used per assay and to check its purity, in particular to verify the absence of contaminating RNA. Secondly, calibration standards were prepared, in order to correct for differences in recovery. The modification levels of these standards were determined by synchronous fluorescence spectrophotometric analysis. Thirdly, the effect on adduct levels of exposure to light during postlabelling was investigated. Exposure of polyaromatic DNA adducts on a PEI-cellulose plate reduced the amounts of adducts detected considerably.