Children sustain high levels of skin DNA photodamage, with a modest increase of serum 25-hydroxyvitamin D3 , after a summer holiday in Northern Europe.

BACKGROUND: Childhood solar ultraviolet radiation (UVR) exposure increases the risk of skin cancer in adulthood, which is associated with mutations caused by UVR-induced cyclobutane pyrimidine dimers (CPD). Solar UVR is also the main source of vitamin D, essential for healthy bone development in children. OBJECTIVES: To assess the impact of a 12-day Baltic Sea (54° N) beach holiday on serum 25-hydroxyvitamin D3 [25(OH)D3 ] and CPD in 32 healthy Polish children (skin types I-IV). METHODS: Blood and urine were collected before and after the holiday and assessed for 25(OH)D3 and excreted CPD, respectively, and personal UVR exposure was measured. Diaries were used to record sunbathing, sunburn and sunscreen use. Before- and after-holiday skin redness and pigmentation were measured by reflectance spectroscopy. RESULTS: The average ± SD daily exposure UVR dose was 2·4 ± 1·5 standard erythema doses (SEDs), which is borderline erythemal. The mean concentration of 25(OH)D3 increased (× 1·24 ± 0·19) from 64·7 ± 13·3 to 79·3 ± 18·7 nmol L-1 (P < 0·001). Mean CPD increased 12·6 ± 10·0-fold from 26·9 ± 17·9 to 248·9 ± 113·4 fmol µmol-1 creatinine (P < 0·001). Increased 25(OH)D3 was accompanied by a very much greater increase in DNA damage associated with carcinogenic potential. Overall, skin type had no significant effects on behavioural, clinical or analytical outcomes, but skin types I/II had more CPD (unadjusted P = 0·0496) than skin types III/IV at the end of the holiday. CONCLUSIONS: Careful consideration must be given to the health outcomes of childhood solar exposure, and a much better understanding of the risk-benefit relationships of such exposure is required. Rigorous photoprotection is necessary for children, even in Northern Europe.

The nucleotide excision repair protein XPC is essential for bulky DNA adducts to promote interleukin-6 expression via the activation of p38-SAPK.

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, and many are potent carcinogens. Benzo[a]pyrene (B[a]P), one of the best-studied PAHs, is metabolized ultimately to the genotoxin anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE triggers stress responses linked to gene expression, cell death and survival. So far, the underlying mechanisms that initiate these signal transduction cascades are unknown. Here we show that BPDE-induced DNA damage is recognized by DNA damage sensor proteins to induce activation of the stress-activated protein kinase (SAPK) p38. Surprisingly, the classical DNA damage response, which involves the kinases ATM and ATR, is not involved in p38-SAPK activation by BPDE. Moreover, the induction of p38-SAPK phosphorylation also occurs in the absence of DNA strand breaks. Instead, increased phosphorylation of p38-SAPK requires the nucleotide excision repair (NER) and DNA damage sensor proteins XPC and mHR23B. Interestingly, other genotoxins such as cisplatin (CDDP), hydrogen peroxide and ultraviolet radiation also enhance XPC-dependent p38-SAPK phosphorylation. In contrast, anti-benzo[c]phenanthrene-3,4-dihydrodiol-1,2-epoxide, the DNA adducts of which are not properly recognized by NER, does not trigger p38-SAPK activation. As a downstream consequence, expression and secretion of the pro-inflammatory cytokine interleukin-6 is induced by BPDE and CDDP in vitro and by CDDP in the murine lung, and depends on XPC. In conclusion, we describe a novel pathway in which DNA damage recognition by NER proteins specifically leads to activation of p38-SAPK to promote inflammatory gene expression.

Transplacental transfer of nitrosodimethylamine in perfused human placenta.

Nitrosodimethylamine (NDMA) is a carcinogenic compound present in tobacco smoke and food such as cured meat, smoked fish and beer. The O(6)-methylguanine formed in human cord blood in mothers highly exposed to such products implicates NDMA exposure of the fetus. Dual recirculating human placental perfusion was used to get direct evidence of the transplacental transfer of NDMA and DNA adduct formation in perfused human placenta. Eleven placentas from normal full-term pregnancies were collected immediately after delivery and an isolated lobule was perfused with 1 or 5 microM of (14)C-NDMA with a reference substance, antipyrine (0.1mg/ml) added to the maternal circulation. Perfusate samples were collected from both maternal and fetal circulations every half an hour for the first two hours and once per hour from thereon. NDMA was analyzed by scintillation counting and antipyrine by high performance liquid chromatography. The transfer of NDMA was comparable to that of antipyrine and probably occurred through passive diffusion, with the concentrations in maternal and fetal sides equilibrating in 2-3h. No indication of any effect by efflux transporters on NDMA kinetics was noticed in the experiments utilizing Caco-2 or MDCK- MDCKII-MDR1 cell culture monolayer in a transwell system, either. Furthermore, no NDMA-DNA-adducts were found after the perfusions and no DNA-binding of NDMA was seen in in vitro incubations with human placental microsomes from 8 additional placentas. Thus, our study demonstrates that the human fetus can be exposed to NDMA from the maternal circulation. According to this study and the literature, NDMA is not metabolized in full-term human placenta from healthy non-smoking, non-drinking mothers. It remains to be studied whether NDMA concentrations high enough to evoke fetal toxicity can be obtained from dietary sources.

32P-post-labelling of 7-(3-chloro-2-hydroxypropyl)guanine in white blood cells of workers occupationally exposed to epichlorohydrin.

Epichlorohydrin (ECH) is a simple 3-carbon epoxide of industrial importance. It has been shown to be genotoxic in several systems and carcinogenic in experimental animals. The aim of the present investigation was to study DNA adducts of ECH as a biomarker of occupational exposure to this chemical. 7-(3-Chloro-2-hydroxypropyl)guanine (7-CHP-guanine) was analysed in DNA from white blood cells using an anion exchange-based adduct enrichment protocol of the (32)P-post-labelling/HPLC-based assay. Blood samples were collected from seven workers handling ECH (exposed), nine workers not handling ECH but normally present in the premises where this chemical is used (potentially exposed) and 13 office and factory workers from locations in the plant where ECH is not handled (controls). 7-CHP-guanine was detected in five of the seven workers exposed to ECH (1.6-7.1 mol/10(9) mol nucleotides) and in two of the nine workers potentially exposed to ECH (0.8-1.5 mol/10(9) mol nucleotides). This adduct was not detected in any of the 13 controls. The difference in adduct levels between exposed workers and controls was statistically significant (Mann-Whitney test, P < 0.001), as was the difference between exposed workers and potentially exposed workers (P = 0.017). The recovery of 7-CHP-guanine in the (32)P-post-labelling assay was on average 48 +/- 7%, which is considerably higher than previously reported for other 7-alkylguanines. The method used had a limit of detection of approximately 0.4 mol adduct/10(9) mol nucleotides using 20 microg DNA. This study shows for the first time ECH-induced DNA adducts in humans and suggests that 7-CHP-guanine may be used as a biomarker of occupational exposure to ECH.

Adducts of acrylonitrile with hemoglobin in nonsmokers and in participants in a smoking cessation program.

Hemoglobin adducts have been used to assess exposure to carcinogenic compounds in tobacco smoke. However, because of background levels in nonsmokers, most adducts that have been studied are not useful for monitoring low-level exposure. Bergmark [(1997) Chem. Res. Toxicol. 10, 78-84] showed that the level of adducts of acrylonitrile (AN) with N-terminal valine (ANVal) increases with increasing cigarette consumption, and the increment from 1 cigarette/day was estimated to be 8 pmol/g of globin. The background level of ANVal in nonsmokers was not quantified (<2 pmol/g of globin). The objective of this study was to determine the background level of ANVal in hemoglobin and to study the stability of this adduct in vivo. Globin samples previously analyzed by Bergmark from 17 nonsmokers and 2 smokers were reanalyzed in the study presented here. Globin samples from 7 additional nonsmokers and from 10 participants in a smoking cessation program were also analyzed. Smoking habits and exposure to environmental tobacco smoke (ETS) were assessed by interview. Only two of the participants completed the program. The levels of ANVal in these 2 subjects decreased after quitting and were at background level by 126 days. The time course of the decrease was compatible with removal of stable adducts. The levels of ANVal in the nonsmokers were 0.76 +/- 0.36 (mean +/- SD) (n = 18; reporting no exposure ETS), 1.1 +/- 0.6 (n = 3; reporting exposure to ETS), and 1. 2 +/- 0.5 pmol/g of globin (n = 3; snuff users). Thus, the adduct level in nonsmokers corresponds to the adduct increment from about 0. 1 cigarette/day. Measurements of the level of ANVal could be used to distinguish between nonsmokers and low-level smokers on an individual level, but larger groups of individuals would be required to detect a possible contribution to the background from passive smoking.

32P-postlabelling of propylene oxide 1- and N(6)-substituted adenine and 3-substituted cytosine/uracil: formation and persistence in vitro and in vivo.

Propylene oxide, a widely used monofunctional alkylating agent, has been shown to be genotoxic in in vitro test systems and induces tumors in the nasal tissues of experimental animals. Propylene oxide, like related alkylating agents, forms several different adducts with DNA bases, but predominantly at the 7-position of guanine. We have previously described the in vitro and in vivo formation and stability of this major adduct. The aim of the present study was to perform a similar investigation of other adducts of propylene oxide. 1-(2-Hydroxypropyl)adenine (1-HP-adenine) and 3-(2-hydroxypropyl)cytosine (3-HP-cytosine), as well as their rearrangement products to N(6)-(2-hydroxypropyl)adenine (N(6)-HP-adenine) and 3-(2-hydroxypropyl)uracil (3-HP-uracil), respectively, were analysed by a very sensitive (32)P-postlabelling method involving nuclease P1 enhancement and radioisotope detector-coupled HPLC separation. All four adducts could be detected in DNA treated in vitro with propylene oxide. The sum of the levels of 1- and N(6)-HP-adenine amounted to 3.5% and the sum of 3-HP-cytosine and 3-HP-uracil to 1.7%, respectively, of 7-(2-hydroxypropyl)guanine (7-HP-guanine). In male Fischer 344 rats exposed to 500 p.p.m. propylene oxide by inhalation for 20 days, 1-HP-adenine was detected in all analysed tissues, including nasal epithelium, lung and lymphocytes, whereas N(6)-HP-adenine was only found in the tissues of the nasal cavities. The highest level of 1-HP-adenine (2.0 mol/10(6) mol of normal nucleotides, i.e. 2% of 7-HP-guanine) was found in the respiratory nasal epithelium, which also represents the major target for tumour induction in the rat following inhalation of propylene oxide. The levels of this adduct in the lung and in the lymphocytes were considerably lower, amounting to 15 and 9%, respectively, of that of the respiratory nasal epithelium. In rats killed 3 days after cessation of exposure, practically no decrease in 1-HP-adenine was observed, indicating no or very slow repair. 3-HP-uracil could only be detected in the respiratory nasal epithelia of propylene-exposed rats and its concentration was as low as 0.02 mol/10(6) mol of normal nucleotides (0.02% of 7-HP-guanine). Since 3-HP-uracil was chemically much more stable than the latter, the obtained animal data suggest repair of the cytosine and/or uracil adducts. Incubation of propylene oxide-reacted DNA with a protein extract from mammalian cells indicated that an enzymatic repair mechanism exists for removal of 3-HP-cytosine, but not for 3-HP-uracil or 1- and N(6)-HP-adenine. Another finding was that uracil glycosylase is probably not involved. The level of 1-HP-adenine in the propylene oxide-exposed rats was approximately 50 times lower than that of 7-HP-guanine. Nevertheless, this adduct is conveniently analysed and has high chemical stability and recovery, which results in high sensitivity (detection limit 0.3 mol/10(9) mol of normal nucleotides using 10 microgram DNA). 1-HP-adenine might, therefore, be considered as an alternative to 7-HP-guanine for monitoring exposure to propylene oxide.

Adducts with haemoglobin and with DNA in epichlorohydrin-exposed rats.

Epichlorohydrin (1-chloro-2,3-epoxypropane; ECH) is an important industrial chemical and a carcinogen in experimental animals. The main aims of the present study were to characterize the adduct formation in female Wistar rats and to identify adducts that could potentially be used in human biomonitoring studies. The total binding of radioactivity to haemoglobin in rats administered 0, 0. 11, 0.22, 0.43, or 0.97 mmol [3H]ECH/kg body weight by i.p. injection, and sacrificed 24 h after treatment, was linearly related to a dose up to 0.43 mmol/kg body weight. The binding at the highest dose was higher than predicted by extrapolation from lower doses, indicating saturation of a metabolic process for elimination of ECH. Ion-exchange chromatography of a globin hydrolysate showed one major radioactivity peak corresponding to S-(3-chloro-2-hydroxypropyl)cysteine. The half-life of this adduct was estimated as about 4 days by analysis of globin from rats administered 0.43 mmol/kg body weight and sacrificed after 1, 2 and 9 days. Crosslinking of the adduct, presumably with glutathione, appeared to be the predominant secondary reaction. Hydrolysis of N-(3-chloro-2-hydroxypropyl)valine, the primary reaction product of ECH with N-terminal valine, would give N-(2,3-dihydroxypropyl)valine. A sensitive gas chromatography/mass spectrometry method for the dihydroxypropyl adduct was used to follow its formation and removal after administration of nonlabelled ECH (0.11 mmol/kg body weight). The level of this adduct reached a maximum of about 20 pmol/g globin after a few weeks, corresponding to about 0.1% of the initial binding of ECH to globin. N-7-(3-Chloro-2-hydroxypropyl)guanine was detected in rats administered 0.97 mmol [3H]ECH/kg body weight and sacrificed 6 h after treatment. The adduct levels in haemoglobin and DNA were compared with previously reported adduct levels in male Fischer 344 rats exposed to propylene oxide. Despite its higher chemical reactivity, the capacity of ECH to alkylate macromolecules in vivo was found to be somewhat lower than that of propylene oxide.

Tissue distribution of DNA adducts in male Fischer rats exposed to 500 ppm of propylene oxide: quantitative analysis of 7-(2-hydroxypropyl)guanine by 32P-postlabelling.

7-(2-Hydroxypropyl)guanine (7-HPG) constitutes the major adduct from alkylation of DNA by the genotoxic carcinogen, propylene oxide. The levels of 7-HPG in DNA of various organs provides a relevant measure of tissue dose. 7-Alkylguanines can induce mutation through abasic sites formed from spontaneous depurination of the adduct. In the current study the formation of 7-HPG was investigated in male Fisher 344 rats exposed to 500 ppm of propylene oxide by inhalation for 6 h/day, 5 days/week, for up to 20 days. 7-HPG was analyzed using the 32P-postlabelling assay with anion-exchange cartridges for adduct enrichment. In animals sacrificed directly following 20 days of exposure, the adduct level was highest in the respiratory nasal epithelium (98.1 adducts per 10(6) nucleotides), followed by olfactory nasal epithelium (58.5), lung (16.3), lymphocytes (9.92), spleen (9.26), liver (4.64), and testis (2.95). The nasal cavity is the major target for tumor induction in the rat following inhalation. This finding is consistent with the major difference in adduct levels observed in nasal epithelium compared to other tissues. In rats sacrificed 3 days after cessation of exposure, the levels of 7-HPG in the aforementioned tissues had, on the average, decreased by about one-quarter of their initial concentrations. This degree of loss closely corresponds to the spontaneous rate of depurination for this adduct (t 1/2 = 120 h), and suggests a low efficiency of repair for 7-HPG in the rat. The postlabelling assay used had a detection limit of one to two adducts per 10(8) nucleotides, i.e. it is likely that this adduct could be analyzed in nasal tissues of rats exposed to less than 1 ppm of propylene oxide.

Propylene oxide: mutagenesis, carcinogenesis and molecular dose.

The results from mutagenic and carcinogenic studies of propylene oxide (PO) and the current efforts to develop molecular dosimetry methods for PO-DNA adducts are reviewed. PO has been shown to be active in several bacterial and mammalian mutagenicity tests and induces site of contact tumors in rodents after long-term administration. Quantitation of N7-(2-hydroxypropyl)guanine (7-HPG) in nasal and hepatic tissues of male F344 rats exposed to 500 ppm PO (6 h/day; 5 days/week for 4 weeks) by inhalation was performed to evaluate the potential of high concentrations of PO to produce adducts in the DNA of rodent tissues and to obtain information necessary for the design of molecular dosimetry studies. The persistence of 7-HPG in nasal and hepatic tissues was studied in rats killed three days after cessation of a 4-week exposure period. DNA samples from exposed and untreated animals were analyzed for 7-HPG by two different methods. The first method consisted of separation of the adduct from DNA by neutral thermal hydrolysis, followed by electrophoretic derivatization of the adduct and gas chromatography-high resolution mass spectrometry (GC-HRMS) analysis. The second method utilized 32P-postlabeling to quantitate the amount of this adduct in rat tissues. Adducts present in tissues from rats killed immediately after cessation of exposure were 835.4 +/- 80.1 (respiratory), 396.8 +/- 53.1 (olfactory) and 34.6 +/- 3.0 (liver) pmol adduct/mumol guanine using GC-HRMS. Lower values, 592.7 +/- 53.3, 296.5 +/- 32.6 and 23.2 +/- 0.6 pmol adduct/mumol guanine were found in respiratory, olfactory and hepatic tissues of rats killed after three days of recovery. Analysis of the tissues by 32P-postlabeling yielded the following values: 445.7 +/- 8.0 (respiratory), 301.6 +/- 49.2 (olfactory) and 20.6 +/- 1.8 (liver) pmol adduct/mumol guanine in DNA of rats killed immediately after exposure cessation and 327.1 +/- 21.7 (respiratory), 185.3 +/- 29.2 (olfactory) and 15.7 +/- 0.9 (liver) pmol adduct/mumol guanine after recovery. Current methods of quantitation did not provide evidence for the endogenous formation of this adduct in control animals. These studies demonstrated that the target tissue for carcinogenesis has much greater alkylation of DNA than liver, a tissue that did not exhibit a carcinogenic response.

32P-Postlabelling of DNA adducts formed by allyl glycidyl ether in vitro and in vivo.

32P-Postlabelling analysis of allyl glycidyl ether-treated DNA after adduct enrichment on anion-exchange cartridges revealed two major and one minor DNA adducts. The major adducts were shown to originate from alkylation at N-7-guanine and N-1-adenine, respectively, while the minor adduct was at N-3-cytosine. In addition, rearrangement products of the 1-adenine and 3-cytosine adducts to N6-adenine and 3-uracil were indicated. The relative amounts of adenine, cytosine and uracil products appeared to be dependent upon conditions (in particular pH) during sample processing and analysis. When nuclease P1 was used for adduct enrichment the adenine, cytosine and uracil adducts, but not the 7-guanine adduct, were detected. The labelling efficiency of the 7-guanine adduct standard was 40-45%. Total recovery of this adduct from allyl glycidyl ether-modified DNA was 9-12%. The labelling efficiency of the 1-adenine adduct standard was 78-82%. Total recovery of this adduct from DNA was approximately 20% when using anion-exchange chromatography for adduct enrichment and 30-34% when using nuclease P1. Preliminary analysis of DNA from mice treated with allyl glycidyl ether indicated 57 times higher level of the 7-guanine adduct, per unit dose, in skin DNA (120 per 10(8) normal nucleotides) after topical application when compared to liver DNA after i.p. administration. The 1-adenine adduct could not be quantified in liver DNA (due to an interfering background product present in untreated animals) and the level of the 3-cytosine adduct was below the detection limit of the method. After topical application the level of the 1 adenine adduct in skin DNA was approximately 30 per 10(8), using either column or nuclease P1 enrichment. The 3-cytosine adduct was detected in skin, but was not quantified.

DNA adduct formation by allyl glycidyl ether.

Glycidyl ethers are reactive epoxides used as components of a variety of epoxy materials. These compounds are known to cause allergic reactions, but since they are generally also genotoxic it would be of interest to evaluate the risk for induction of such effects. Reaction products of allyl glycidyl ether with nucleic acid components were therefore studied. Adduct standards of expected major products in DNA were prepared and assigned to N-7-guanine, N-1- and N-3-adenine and N-3-cytosine. The adducts were characterized by UV spectroscopy, and the adduct to N-1-adenine also by mass spectrometry and nuclear magnetic resonance spectroscopy. In analogy with the formation of corresponding reaction products of other simple epoxides the N-1-adenine adduct rearranged in a base catalysed reaction to N6 and the N-3-cytosine adduct deaminated to form the corresponding N-3-uracil adduct. For allyl glycidyl ether these further reactions of the N-1-adenine and N-3-cytosine adducts were, however, slower than has been observed for corresponding products of other epoxides, but faster than for methylated and ethylated products. In double-stranded salmon testis DNA treated in vitro with allyl glycidyl ether, the major product was found at N-7-guanine, followed by those at N-1-adenine, N-3-adenine and N-3-cytosine (including N-3-uracil). A minor amount of an N6-adenine adduct was also detected, but only after 48 h of reaction. In single-stranded DNA the yield of the N-1-adenine adduct was increased to about the level of the N-7-guanine adduct. The level of the N-3-cytosine adduct was also considerably higher in single-stranded DNA and was the third largest adduct. The reactivity of N-3-adenine was decreased in single-stranded DNA and since other adducts increased the relative yield of this adduct was very low. The N-7-guanine and N-3-adenine adducts were lost from DNA as a consequence of depurination with half-lives in double-stranded DNA at 37 degrees C and pH 7.4 of 38 and 20 h, respectively. The rates of losses (due to depurination or rearrangement) of initially formed adducts in DNA increased in the order N-1-adenine < N-7-guanine approximately N-3-cytosine < N-3-adenine and were faster in single- than in double-stranded DNA. Taking only the rate of formation and chemical stability into consideration, the adducts with N-1-adenine and N-7-guanine seem to be the most promising candidates for monitoring allyl glycidyl ether exposures in vivo.

Oxazolidonylethyl adducts to hemoglobin and DNA following nornitrogen mustard exposure.

Formation of adducts to hemoglobin (Hb) and DNA of nornitrogen mustard (NNM) was studied with the aim of developing a method for monitoring exposure to NNM. Adducts to N-terminal valines in Hb were studied by the N-alkyl Edman method using pentafluorophenyl isothiocyanate (PFPITC) as the derivatizing reagent. In preliminary studies five major Hb adducts were shown to be formed in reaction of NNM with red cell hemolysate in vitro. Following treatment with PFPITC three of these were found to be pentafluorophenylthiohydantoins (PFPTHs) of N-alkylated valines and the fourth probably originates from NNM esters in which PFPITC had reacted with the nitrogen of N-chloroethylaminoethyl. A PFPTH was found to originate from N-2-(3-oxazolidonyl)ethylvaline, Val-OZ. Val-OZ is formed in reaction, with ring closure to oxazolidone, of CO2 with the 2-chloroethylamino group in the primary valine-N adduct. Besides a few other adducts, Val-OZ was also observed in mouse Hb following injection of NNM, and also after injection of cyclophosphamide. Following reaction in vitro of NNM with DNA, three major adducts to guanine-N-7 were observed; one of them, 7-(N'-(2-chloroethyl)-2-aminoethyl]-guanine (NNMCl), was converted by carbonate to 7-(2-3-oxazolidonyl)ethyl]guanine (Gua-OZ). In mice treated with NNM, Gua-Oz was the only DNA adduct observed. Val-Oz is a chemically stable Hb adduct, potentially useful for monitoring exposures to NNM and cyclophosphamide.

Formation of N-7-(2-carbamoyl-2-hydroxyethyl)guanine in DNA of the mouse and the rat following intraperitoneal administration of [14C]acrylamide.

Acrylamide is an alkylating agent which reacts very slowly in direct reactions with DNA and is negative in the Ames test, but is carcinogenic in mice and rats. In order to explain the cancer-initiating properties of acrylamide we have studied DNA adduct formation in vitro with a metabolizing system and in vivo in mice and rats following i.p. administration of 14C-labeled acrylamide. A major adduct found in both species was N-7-(2-carbamoyl-2-hydroxy-ethyl)guanine, formed by reaction of the DNA with the epoxide metabolite glycidamide. The levels of this adduct were similar in the different organs of the two rodent species, which supports the notion that glycidamide is relatively evenly distributed among tissues and that the organ-specificity in acrylamide carcinogenesis cannot be explained by a selective accumulation of the DNA-reactive metabolite in target organs.

In vivo tissue dosimetry as a basis for cross-species extrapolation in cancer risk assessment of propylene oxide.

The potential for causing carcinogenic and mutagenic effects has been the main concern when assessing the risks associated with low-level exposures of humans to the industrially important epoxide, propylene oxide (PrO). For regulatory purposes, surface-based extrapolation has been used to determine the human equivalent dose from cancer data obtained in rodents. In this context the tissue dose will more adequately reflect inter- and intraspecies differences with respect to pharmacokinetic parameters than is the case for conventional representations of exposure. The formation of adducts in nucleophilic molecular targets by directly acting electrophilic agents, like epoxides, is thought to be closely linked to the process of cancer initiation. To investigate whether tissue dose is correlated to surface area of the exposed organism, the in vivo adduct levels in hemoglobin and DNA have been determined in mice, rats, and dogs after exposure to PrO by injection as well as by inhalation. The results obtained indicate that the dose in blood is virtually the same in the three investigated animal species, whereas surface-area based extrapolation predicts a difference by a factor of about seven between the mouse and the dog. Although the data base is more limited, this conclusion is also supported by measurements of DNA alkylation is selected tissues. The variations actually observed are not related to the surface area of the animal. No significant differences could be found between administration of PrO by injection or by inhalation. For this reason, the surface-based extrapolation model for estimation of the human equivalent dose is not appropriate, and the carcinogenic potency factors for PrO as previously derived by the U.S. EPA should probably be revised downward by a factor of 10 to 13.

Comparative two-stage cancer tests of ethylene oxide, N-(2-hydroxyethyl)-N-nitrosourea and X-rays.

Mutagenicity tests have shown that the potencies of ethylene oxide and other alkylating agents relative to that of low-LET ionising radiation are approximately the same in different biological systems. In the present study this relationship, the radiation-dose equivalence ("rad-equivalence") of doses of genotoxic chemicals, was tested for the induction of tumours in skin and lung of mice using different initiation-promotion protocols. The initiators used were X-rays, ethylene oxide and N-(2-hydroxyethyl)-N-nitrosourea (HOENU). This short-term treatment was followed by treatment with the "promoters" 12-O-tetradecanoylphorbol 13-acetate (TPA) and carbon tetrachloride. Unexpectedly, the animals treated with carbon tetrachloride did not show treatment-related liver tumours, but exhibited precocious death, mostly with lung tumours. According to estimates from in vitro tests the total in vivo dose from exposure to 400 ppm for 4 x 5 h corresponds to 700 rad-equivalents. Although still with great statistical uncertainty, this ratio is supported by the observed time-dependent frequencies of animals with papillomas (in the TPA series) and lung tumours (in the carbon tetrachloride series). Animals treated with HOENU exhibited high incidences of tumours of both these types in approximate agreement with the higher rad-equivalence of the treatments with this compound.

DNA adducts among personnel servicing and loading diesel vehicles.

The levels of aromatic DNA adducts were compared by the 32P-postlabeling assay between the lymphocytes isolated from bus maintenance and truck terminal workers, using hospital mechanics as a control group. The adduct levels were elevated in all the bus and truck terminal workers. Within the bus maintenance personnel, garage workers had the highest levels of adducts. Within the terminal workers those driving diesel forklifts had the highest adduct levels. The results suggest that diesel exhaust contributes to the level of adducts.

Recoveries of DNA adducts of polycyclic aromatic hydrocarbons in the 32P-postlabelling assay.

The 32P-postlabelling assay for analysis of DNA adducts of chemical carcinogens has been applied in a large number of experimental animal and human studies. Most human studies have dealt with occupational and environmental exposures to polycyclic aromatic hydrocarbons (PAHs). The postlabelling assay does not allow direct chemical identification, and most studies with this method have not been performed in a quantitative way. Very little is therefore known about the identity and absolute levels of adducts, which are important contributors to the process of risk identification and quantitation. In the present study it was, therefore, decided to test some parameters suspected to affect recoveries of adducts in the phosphorylation step of the assay. For this purpose 12 different PAHs were reacted individually and in a mixture with DNA in the presence of a rat liver S9 metabolizing system. Different concentrations of ATP, calcium chloride and polynucleotide kinase were tested using the nuclease P1 enhancement. We found that each factor contributed to adduct recovery and that optimal conditions could be defined. Diluting the modified DNA samples up to 1000 times had little influence on the recoveries of adducts. Comparing the nuclease P1 and the butanol extraction procedures for adduct purification showed that both methods gave similar patterns and levels of major adducts. The absolute recoveries in postlabelling, based on 3H-binding of radiolabelled compounds, were for most of the tested compounds relatively low. The fact that the nuclease P1 and the butanol extraction procedures gave similar recoveries points towards common factor(s) involved in the reduction of the recovered adduct levels. Based on the observed recoveries the conclusion can be drawn that when postlabelling related adducts in human samples the true total adduct levels can be considerably underestimated, even if optimal conditions are used.

32P-postlabelling analysis of DNA adducts of 4,4'-methylenebis(2-chloroaniline) in target and nontarget tissues in the dog and their implications for human risk assessment.

4,4'-Methylenebis(2-chloroaniline) (MOCA) has considerable human occupational exposure and it induces urinary bladder tumors in the dog, a species that has been often used as a model for aromatic amine-induced urinary bladder carcinogenesis in humans. Metabolic activation and formation of DNA adducts are considered to be critical steps in this process; and two major C8-adenine adducts have been shown to be formed in vitro by reaction with the proximate carcinogenic metabolite N-hydroxy-MOCA. MOCA-DNA adducts have also been detected in vivo in treated rats and in exfoliated urothelium of a worker accidentally exposed to MOCA. Thus, the aim of this study was to detect and quantify DNA adducts in the urinary bladder of dogs exposed to MOCA and thereby provide data that could be useful for risk assessment after human exposure to MOCA. Beagle dogs were treated with single and multiple doses of MOCA and DNA adduct levels were determined in liver and bladder epithelium. After a single dose, adduct levels in the liver were 1.5-fold higher than that in the bladder epithelium. Adduct levels in these two organs increased 3- to 5-fold after 10 doses and adducts in the liver were then 2.8-fold higher than that in the bladder epithelium. The levels found in these two organs after single exposures were compared, per unit exposure dose, with that reported for other carcinogenic aromatic amines. The comparison showed that MOCA was as effective in DNA adduct formation as most other potent urinary bladder carcinogens. These results suggest that MOCA may have high carcinogenic potential in humans and are consistent with the recent classification of MOCA as a probable human carcinogen.