In vitro cytotoxicity of chrysotile asbestos to human pulmonary alveolar macrophages is decreased by organosilane coating and surfactant

Human pulmonary alveolar macrophages were used to quantitate the cytotoxic effect of surface-altered chrysotile asbestos. Little difference was observed in mortality between chrysotile asbestos that was surface-treated to a 42% extent by a hydrophobic organosilane or untreated chrysotile. Little or no effect on mortality was observed when human pulmonary alveolar macrophages were cultured with untreated chrysotile or acid-leached asbestos in the presence of 10 mM dipalmitoyl lecithin. However, when human pulmonary alveolar macrophages were cultured with a hydrophobically-treated (to a 42% or 95% extent) chrysotile asbestos in the presence of 10 mM dipalmitoyl lecithin, a statistically significant decrease in mortality was observed compared to untreated chrysotile. No mutagenic activity was observed when V79 cells were cultured with acid-leached, or 42% hydrophobically-treated chrysotile asbestos, even when human pulmonary alveolar macrophages were included as an activation source. The 95% hydrophobically-treated and acid-leached chrysotile also exhibited decreased binding of benzo[a]pyrene compared to untreated chrysotile asbestos.Abbreviations AHH aryl hydrocarbon hydroxylase - B(a)P benzo[a]pyrene - CA chrysotile asbestos - CHO Chinese hamster ovary - DL dipalmitoyl lecithin - DMEM Dulbecco's Modified Eagle's Medium - FBS fetal bovine serum - O^r resistance to ouabain - PAH polycyclic aromatic hydrocarbon - PAM pulmonary alveolar macrophage - SCE sister chromatid exchange Deceased.     

Simultaneous detection of DNA strand breaks and unscheduled DNA synthesis in mutagen-treated human lymphocytes in the absence of hydroxyurea

Human lymphocytes in the quiescent state were exposed to UVC radiation. After irradiation the cells were allowed to repair for various times in the presence of [3H]thymidine or [3H]deoxycytidine in the culture medium. Hydroxyurea was not used to suppress semiconservative DNA replication in the small number of growing cells. After incubation DNA strand breaks were detected by the DNA-unwinding method and the amount of 3H incorporation in DNA was measured by liquid scintillation counting. The results show that the yield of DNA strand breaks and the amount of unscheduled DNA synthesis (UDS) can be measured from the same lymphocyte sample. A low background 3H incorporation in untreated cells could be achieved even in the absence of hydroxyurea. This requires, however, that 3H incorporation is measured only in the double-stranded DNA and that [3H]dCyd is used instead of [3H]dThd as the labelled deoxynucleoside.     

Vitronectin adsorption to chrysotile asbestos increases fiber phagocytosis and toxicity for mesothelial cells

Biological modification of asbestos fibers can alter their interaction with target cells. We have shown that vitronectin (VN), a major adhesive protein in serum, adsorbs to crocidolite asbestos and increases fiber phagocytosis by mesothelial cells via integrins. Because chrysotile asbestos differs significantly from crocidolite in charge and shape, we asked whether VN would also adsorb to chrysotile asbestos and increase its toxicity for mesothelial cells. We found that VN, either from purified solutions or from serum, adsorbed to chrysotile but at a lower amount per surface area than to crocidolite. Nevertheless, VN coating increased the phagocytosis of chrysotile as well as of crocidolite asbestos. VN coating of both chrysotile and crocidolite, but not of glass beads, increased intracellular oxidation and apoptosis of mesothelial cells. The additional apoptosis could be blocked by integrin-ligand blockade with arginine-glycine-aspartic acid peptides, confirming a role for integrins in the fiber-induced toxicity. We conclude that VN increases the phagocytosis of chrysotile as well as of crocidolite asbestos and that phagocytosis is important in fiber-induced toxicity for mesothelial cells.     

Unscheduled DNA synthesis induced in mouse spermatids after combined treatment with methyl methanesulfonate and X-rays.

Unscheduled DNA synthesis (UDS), which is considered to be DNA repair, has been studied in early- to mid-spermatid stages of the mouse after combined treatments with X-rays and methyl methanesulfonate (MMS). UDS in spermatids was detected by giving testicular injections of [methyl-3H]thymidine ([3H]dThd) and making use of the fact that no scheduled DNA synthesis occurs in the germ cells after the last S period in primary spermatocytes. X-rays and MMS are each able to induce UDS in mouse spermatids. However, there was a statistically significant reduction in the amount of UDS observed when X-ray exposures of from 200 to 600 R were given 4 h before an i.p. injection of 75 mg/kg of MMS and concurrent testicular injections of [3H]dThd. This reduction in UDS is more than can be explained by the completion of repair of X-ray-induced DNA lesions. We suggest that the reduction in UDS is the result of an X-ray-produced impairment of a least a part of the repair mechanism involved in correcting MMS-induced DNA lesions. When the time interval between a 600-R X-ray exposure and MMS treatment was between 3 and 20 h (latest time interval s;udied) there was a statistically significant reduction of UDS in the spermatids. No significant decrease in UDS response occurred when the time interval between radiation exposure and MMS treatment was less than approximately 3 h.     

A UV-sensitive human clonal cell line,RSa, which has low repair activity

The repair activity of a human transformed cell line, RSa, which was found to be highly sensitive to the lethal effects of 254 nm far-ultraviolet radiation, was compared with that of HeLa cells by evaluating the range of UV-induced incorporation of [methyl-³H]thymidine ([³H]dThd) or 5-[6-³H]bromodeoxyuridine ([³H]BrdUrd) into deoxyribonucleic acid. Direct scintillation counting was used for measuring the extent of unscheduled DNA synthesis (UDS) in UV-irradiated cells, which were treated with hydroxyurea or with arginine deprivation. More quantitative measurements were made by using the density labeling and equilibrium centrifugation method for assaying repair replication. All the amounts of UDS and repair replication in RSa cells were markedly below those in HeLa cells. The possible relationships of the low repair activity to abnormally high UV sensitivity in RSa cells are discussed.     

Induction of alkaline-labile sites in DNA by benzo [a]pyrene and the repair of those lesions in cultured Chinese hamster cells

Formation of alkaline-labile sites in DNA by S9-activated benzo [a]pyrene (B [a]P) and the repair of those lesions were investigated using the technique of alkaline elution in cultured Chinese hamster V79 cells. When the cells were treated with B [a]P (1-5 micrograms/ml) there was negligible increase in DNA elution at pH 12.1 as compared to untreated controls. However, the elution of DNA increased at pH 12.6 with a concentration dependency, thereby indicating formation of alkaline-labile sites in DNA by B [a]P. After 4 h of repair incubation the elution of DNA at pH 12.6 of B [a]P (5 micrograms/ml) treated cells returned to the control levels. The half-life of alkaline-labile sites formed by B [a]P was approximately 1.5 h. Inhibitors of DNA-repair synthesis, hydroxyurea (HU) and 1-beta-D-arabinofuranosyl cytosine (ara-C) when added simultaneously with S9-activated B [a]P for 3 h showed an increase in elution of DNA at pH 12.1, indicating that a population of B [a]P-induced DNA lesions could be removed by a rapid DNA-repair process. These results indicate that at least two kinds of DNA lesions, repairable alkaline-labile sites and rapidly repairable DNA single-strand breaks, are detected after B [a]P treatment by the use of the alkaline elution procedure, by changing elution pH.     

Asbestos-elicited catecholamine secretion from isolated bovine adrenal chromaffin cells

Standard (UICC) chrysotile B asbestos fibres caused rapid (within minutes) 5-to-8-fold stimulations of catecholamine secretion from isolated bovine adrenal chromaffin cells without affecting their viability (97%). The stimulation of catecholamine secretion by asbestos was selective to chrysotile type fibres, half-maximal stimulation by standard chrysotile B, chrysotile A, crocidolite, amosite and silica fibres being observed at 7, 73, 160, 250 and ? 500 μg per ml, respectively. The secretory effect of chrysotile B was additive to that of acetylcholine and blocked by either the divalent cations, Co²⁺, Ni²⁺ and Mg²⁺ or the ion chelators, EGTA and EDTA. Conversely, neither verapamil, methoxyverapamil, or removal of extracellular calcium affected the asbestos-evoked catecholamine secretion. These data indicate that the selective stimulatory effect of chrysotile type asbestos on adrenal chromaffin cells can be mediated by membrane or intracellular calcium and raise the question of the possible involvement of catecholamines in the pathogenesis of asbestos related diseases.     

Modulation of genotoxic effects in asbestos-exposed primary human mesothelial cells by radical scavengers, metal chelators and a glutathione precursor

The genotoxicity of asbestos fibers is generally mediated by reactive oxygen species (ROS) and by insufficient antioxidant protection. To further elucidate which radicals are involved in asbestos-mediated genotoxicity and to which extent, we have carried out experiments with the metal chelators deferoxamine (DEF) and phytic acid (PA), and with the radical scavengers superoxide dismutase (SOD), dimethylthiourea (DMTU) and the glutathione precursor Nacystelyn trade mark (NAL). We investigated the influence of these compounds on the potency of crocidolite, an amphibole asbestos fiber with a high iron content (27%), and chrysotile, a serpentine asbestos fiber with a low iron content (2%), to induce micronuclei (MN) in human mesothelial cells (HMC) after an exposure time of 24-72 h. Our results show that the number of crocidolite-induced MN is significantly reduced after pretreatment of fibers with PA and DEF. This effect was not observed with chrysotile. In contrast, simultaneous treatment of cells with asbestos and the OH*scavenging DMTU or the O2- -scavenging SOD significantly decreased the number of MN induced by chrysotile and crocidolite. In particular, DMTU almost completely suppressed micronucleus induction by both fiber types. A similar effect was observed in the presence of the H(2)O(2)-scavenging NAL after chrysotile treatment of HMC. By means of kinetochore analysis, it could be shown that the number of clastogenic events is decreased after PA and DEF pretreatment of fibers as well as after application of the above-mentioned scavengers. Our results show that chrysotile asbestos induces an increased release of H(2)O(2) in contrast to crocidolite. Also, the iron content of the fiber plays an important role in radical formation, but nevertheless, chrysotile produces oxy radicals to a similar extent as crocidolite, probably by phagocytosis-mediated oxidative bursting.     

The autoradiographic test for unscheduled DNA synthesis: a sensitive assay for the detection of DNA repair in the HepG2 cell line

We assessed the DNA-repair capacity of HepG2 cells, which were derived from a human hepatoma, by the unscheduled DNA synthesis assay, using the autoradiography protocol (UDS-AR). We evaluated DNA repair following exposure to direct mutagens (4-nitroquinoline-N-oxide (4-NQO), methyl methanesulfonate (MMS)), to mutagens requiring metabolic activation (benzo[a]pyrene (B[a]P), 2-acetylaminofluorene (2-AAF), N-dimethylnitrosoamine (NDMA)) or to structurally related non-mutagens such as pyrene and 4-acetylaminofluorene (4-AAF). All positive compounds tested induced UDS in HepG2 cells. With 4-NQO and MMS, a concentration-dependent increase in net nuclear grains per cell was observed, with 73 and 90% of cells, respectively, in repair at the highest concentration. B[a]P, 2-AAF and NDMA displayed similar dose-dependent UDS responses, but the percentage of cells in repair was lower (about 45%) than that for 4-NQO and MMS. We assessed the genotoxicity of the compounds tested by determining IC(5NNG): the concentration required to induce 5NNG. The compounds studied were ranked in order of IC(5NNG) as follows: 4-NQO = B[a]P > 2-AAF > MMS > NDMA. The UDS assay discriminated between mutagens and non-mutagens, as pyrene and 4-AAF failed to induce DNA repair. The present study demonstrates that UDS can be used as an endpoint for the detection of DNA damage in HepG2 cells.     

Asbestos catalyzes the formation of the 6-oxobenzo[a]pyrene radical from 6-hydroxybenzo[a]pyrene

Crocidolite asbestos catalyzes the oxidation of 6-hydroxybenzo[a]pyrene, a metabolite of benzo[a]pyrene, to the 6-oxobenzo[a]pyrene radical as determined by electron spin resonance spectroscopy. This may be a mechanism whereby inhaled asbestos enhances the incidence of lung cancer induced by cigarette smoke, which contains benzo[a]pyrene.     

Inability of chrysotile asbestos fibers to modulate the 2-acetylaminofluorene-induced UDS in primary cultures of rat hepatocytes

There is now growing evidence that asbestos fibers could act in association with genotoxic compounds, either as cocarcinogens or promoters, in the process of carcinogenesis. The hepatocyte/UDS assay system has been taken to advantage to investigate the capacity of fibers to modulate the effects of genotoxic compounds on the cell, as we previously demonstrated the hepatocytes can engage in phagocytosis of chrysotile fibers. Measurement of UDS was performed by a biochemical procedure involving liquid scintillation counting (LSC) of a purified DNA fraction as well as by radioautography. Both LSC and radioautography revealed that chrysotile asbestos fibers UICC B at concentrations up to 100 micrograms/ml do not elicit UDS, whereas 2-acetylaminofluorene (2-AAF) at low concentrations (0.05-0.625 micrograms/ml) significantly induces it in parallel positive controls. In an attempt to test the cocarcinogen hypothesis, cultures of hepatocytes were simultaneously exposed for 20 h to 2-AAF (0.05 and 0.25 micrograms/ml) and asbestos fibers (1 and 10 micrograms/ml) given as simple mixtures. It was found that the 2-AAF-induced UDS activity was the same whether fibers were present or not. This was observed with both UDS evaluation procedures at all concentration combinations selected. An analysis of variance applied to the data collected from several experiments confirmed that there was no significant 2-AAF-fiber interaction. Our data suggest the absence of intrinsic genotoxic properties for chrysotile fibers. They also indicate that the modulation of the cellular response to genotoxic agents by asbestos fibers is not detected under our test conditions and may require longer-term exposures to be expressed.     

Reversible permeabilization of lymphocytes destroys the incorporation of deoxythymidine but not of deoxycytidine

The total uptake, phosphorylation and incorporation of thymidine (dThd) and deoxycytidine (dCyd) were compared in intact and reversibly permeabilized human tonsillar lymphocytes. The total uptake of [3H]dThd was lower than that of [5-3H]dCyd, but almost all of [3H]dThd was incorporated into DNA. However, the main part of [5-3H]dCyd taken up by the lymphocytes was found in the pool as phosphorylated nucleoside (55%), and only a smaller part (13%) was incorporated into DNA. Phosphorylated nucleosides were determined by DEAE-cellulose sheets in the ethanol-soluble fraction of the cells. The reversible permeabilization of lymphocytes by Dextran T-150 destroys totally the [3H]dThd incorporation, while [5-3H]dCyd incorporation decreased only to 60% of intact cells. During permeabilization the phosphorylation of both nucleosides increased severalfold. After permeabilization all [3H]dThd was in dTMP form, while [5-3H]dCyd was also found in dCDP (3%) and dCTP (38%) form. In the meanwhile, 22% of thymidine kinase, 63% of deoxycytidine kinase and 98% of DNA polymerase activity were measured in permeabilized cells as compared to intact cells. The results suggest different relationships between the lymphocyte plasma membrane and the salvage pathways of the two pyrimidine nucleosides.     

Cytokine and free radical responses of alveolar macrophages in vitro to asbestos fibres

A method for culturing primary rat alveolar macrophages (AMs) for 14 days was used to compare their responses to crocidolite and chrysotile asbestos fibres. Exposure to crocidolite increased production of tumour necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta), whereas exposure to chrysotile did not; neither fibre altered the production of interleukin 6 (IL-6). IL-1beta production increased progressively, while TNF-alpha was fully elevated from day 1. Conversely, chrysotile, but not crocidolite, increased production of superoxide anion and nitric oxide (NO) radicals. These differential responses were only observed by extending the culture beyond the usual 1-3 days.     

Repair of daughter strand gaps in nascent DNA from mouse epidermal cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

Alkaline sucrose gradient analysis of [methyl-3H]thymidine-pulse-labeled DNA was used to study the effect of (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide I), a potent mutagen and carcinogen, and (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene-diol epoxide II), a weaker mutagen and carcinogen, on the size of newly synthesized DNA in primary cultures of mouse epidermal cells. Both isomers caused a dose-dependent decrease in the size of newly synthesized DNA and in the rate of [methyl-3H]thymidine incorporation into DNA. When the pulse time was increased in the treated cells so that the amount of [methyl-3H]thymidine incorporation was equal to the control, newly synthesized DNA from exposed cells was still considerably smaller than DNA from control cells. The low molecular weight of the nascent DNA from treated cells was consistent with, but not indicative of, the presence of gaps in the nascent DNA from the treated cells. Evidence of gapped DNA synthesis was obtained by treatment of extracted DNA with a single-strand specific endonuclease from Neurospora crassa. The endonuclease treatment did not significantly alter the profile of [methyl-3H]thymidine prelabeled DNA from benzo[a]pyrene-diol epoxide-treated cultures but did introduce double-stand breaks in pulse-labeled DNA from treated cultures. The numbers of [14C]benzo[a]pyrene-diol epoxide I or [3H]benzo[a]pyrenediol epoxide II-DNA-bound adducts and daughter strand gaps were compared at several dose levels. Treatment with either isomer yielded one gap in the nascent DNA/DNA-bound adduct. Pulse-chase experiments showed that gaps in the nascent DNA were closed with time.     

Comparative induction of unscheduled DNA synthesis by physical and chemical agents in non-proliferating primary cultures of rat hepatocytes

The incorporation of [³H]thymidine into DNA due to unscheduled DNA synthesis (UDS) induced by N-OH-2-acetylaminofluorene (N-OH-AAF), aflatoxin B₁ (AFB₁), ethyl methanesulfonate (EMS) and ultra-violet light was quantitated by autoradiography and by scintillation spectrometry on acid precipitable macromolecules or DNA insolated by isopycnic banding in cesium chloride (CsCl). Dose-dependent increases in UDS due to N-OH-AAF and AFB₁ treatment were found. Only 2-fold increases at the highest dose levels were found, however, when incorporated [³H]thymidine was quantitated by scintillation spectrometry. Seven, 11, and 25-fold increases in UDS induced by AFB₁, N-OH-AAF and ultra-violet light, respectively, were found when incorporated [³H]thymidine was quantitated by autoradiography, indicating a high sensitivity for detecting ‘long patch’ repair by this technique. Scintillation spectrometry was completely ineffective in detecting EMS-induced UDS, whereas autoradiography demonstrated a small, but significant induction in [³H]thymidine incorporation at high dose levels. The non-proliferative nature of the primary hepatocyte prohibits the uniform radioactive prelabeling of DNA, necessary in other techniques, for the detection of ‘short patch’ repair induced by compounds such as EMS. Therefore, the sensitivity of the primary cultured rat hepatocyte in conjunction with UDS for detecting DNA damage caused by mutagens and carcinogens which induce ‘short patch’ repair may be limited to the autoradiographic analysis of the unscheduled incorporation of [³H]thymidine.     

Comparative study, with photoelectron spectroscopy of the adsorption of phospholipids by 3 fibrous silicate minerals (chrysotile, crocidolite, attapulgite)

The method used in this study for preparing mineral samples has allowed us to obtain a highly reproducible surface state, which in turn means that we can better exploit the results of the XPS analysis. With high initial concentrations, the three minerals studied appeared to be covered with a bilayer of phospholipids. The greatest adsorption was obtained with initial phospholipid concentrations of 100, 150 and 300 micrograms/ml for crocidolite, chrysotile and attapulgite respectively. It would appear that these differences in surface reactivity can be related to the different levels of risk associated with the inhalation of these fibres.     

Diethyldithiocarbamate inhibits scheduled and unscheduled DNA synthesis of rat thymocytes in vitro and in vivo--dose-effect relationships and mechanisms of action

In vitro as well as in animal models, diethyldithiocarbamate (DDC) modifies the tumoricidal activity of some antineoplastic agents. To gain further information about the mechanism of action of DDC, we measured (i) in vitro and (ii) in vivo changes in DNA synthesis of rat thymocytes. (i) In vitro, the scheduled (SDS) and unscheduled (UDS) incorporation of [3H]thymidine ([3H]dT) into DNA of rat thymic cells were biphasically inhibited in a dose range of 1-1000 micrograms DDC/ml. The UV-induced UDS was totally suppressed by 10 and 100 micrograms DDC/ml. (ii) In vivo, 1-4 h following intraperitoneal administration of 250-1000 mg DDC per kg body wt., SDS and UDS were inhibited up to about 80% in a dose-dependent manner. Nucleoid sedimentation, uptake of [3H]dT into the cells, and the pattern of phosphorylation of the intracellular [3H]dT following DDC treatment did not reveal any differences to the controls. A possible effect of DDC treatment on the ribonucleotide reductase and the DNA polymerase alpha is suggested.     

The effect of asbestos cement, UICC asbestos samples and quartz on the peritoneum of the mouse.]

Aqueous suspensions of asbestos cement powder injected experimentally into the peritoneal cavity of mice act as a fibrogenic agent, as do chrysotile asbestos or chrysotile asbestos-containing soil samples. The fibrotic nodules caused by the dust resemble morphologically silicosis granulomas. In addition, asbestos cement has a characteristically strong cytotoxic effect during the first 2 weeks of the experiment. It is suggested that this is due to the chrysotile asbestos and/or the calcite component of the powder. Amosite and crocidolite, on the other hand, induce a diffuse peritoneal fibrosis with the appearance of numerous foreign body giant cells and asbestos bodies. Dust particles displaced to the regional lymph nodes are frequent in the animals treated with quartz, asbestos cement and asbestos-containing soil samples. A spindle cell type sarcoma arising from the visceral peritoneum is observed in animals injected with crocidolite or asbestos cement. In addition, dusts containing chrysotile asbestos induce considerable amyloidosis of the liver and spleen.     

Bacterial mutagenicity of two cyclopentafused isomers of benzpyrene

Two novel cyclopentafused polycyclic aromatic hydrocarbons, naphtho(1,2,3-mno)acephenanthrylene (cyclopenta benzo[e]pyrene) and naphtho(2,1,8-hij)acephenanthrylene (cyclopenta(ij)benzo[a]pyrene) were evaluated for mutagenic activity in the Ames Salmonella typhimurium plate incorporation assay. Both compounds required S9 metabolic activation, and showed optimal activity at low S9 concentrations (below 0.6 mg/plate). Both compounds induced frameshift and base-pair substitution mutations, being active in strains TA98, TA100, TA1537, TA1538 and TA104, but not in strain TA1535. Cyclopenta(ij)benzo[a]pyrene was more active than cyclopentabenzo[e]pyrene, and both were more potent than their parent ring systems, benzo[a]pyrene and benzo[e]pyrene, respectively. Cyclopenta(ij)benzo[a]pyrene was more active in strain TA104 than in TA100 or TA98 (250-470, 340 and 80-100 rev/nmole) as was benzo[a]pyrene (120, 70 and 40 rev/nmole respectively); cyclopentabenzo[e]pyrene was more active in TA100 than TA104 or TA98 (70 versus 50 and 40 rev/nmole), and benzo[e]pyrene showed a similar pattern (4, 3.5 and 0.6 rev/nmole). The relative potencies of the four compounds are in accord with predictions based on perturbational molecular orbital calculations. The peak of activity at low S9 concentrations is consistent with epoxidation at the cyclopentafused ring being the major route of metabolic activation for both these cyclopentafused compounds.     

Metabolism and binding of benzo[a]pyrene in randomly-proliferating,confluent and s-phase human skin fibroblasts

The metabolism of benzo[a]pyrene in randomly proliferating and confluent cultures of human skin fibroblast cells was compared with cell cultures in early S phase of the cell cycle after a G1 block. When each cell population was exposed to [G-³H]benzo[a]pyrene for 24 hours and the organic soluble metabolites in the extracellular medium and intracellular components were analyzed by HPLC, a quantitative increase in metabolism was observed in the confluent cell populations. The amount of organic soluble metabolites in the extracellular medium of the confluent dense cultures was 2.7 times the amount found in randomly proliferating cultures and 1.5 times that of the synchronized cultures. The trans-7,8- and 9,10 dihydrodiols and 3-hydroxy benzo[a]pyrene were the major metabolites formed. Small amounts of the sulphate conjugate, 9-hydroxy-benzo[a]pyrene and the tetrols were also detected. Cytoplasmic as well as nuclear extracts from the confluent cell cultures also contained higher amounts of metabolites compared to those from the randomly proliferating and S-phase cells. The levels of DNA modification by metabolically activated benzo[a]pyrene did not differ among the randomly proliferating, confluent and S-phase cells. However, the S-phase cells exhibited approximately 50-fold increase in the frequency of transformation compared to the randomly proliferating cells. Confluent cells were not transformed by benzo[a]pyrene. These data suggest that factors other than random modification of DNA by the carcinogen might have a significant role in the expression of a transformed phenotype and that metabolism and transformation are not directly related. Furthermore, confluent dense cultures with a heightened capability for metabolism of benzo[a]pyrene were more active in the detoxification of benzo[a]pyrene than in the production of the metabolites associated with cellular transformation.Abbreviations BaP benzo[a]pyrene - BaP-4,5-diol trans-4,5 dihydroxy-4,5-dihydrobenzo[a]pyrene - BaP-7,8-diol trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene - Bap-9,10-diol trans-9,10-dihydroxy-9,10 dihydrobenzo[a]pyrene - CM complete medium - HNF human neonatal foreskin - HPLC high pressure liquid chromatography - PAH polycyclic aromatic hydrocarbon - PDL population doubling - RP randomly proliferating