Familial bladder cancer in the National Swedish Family Cancer Database.

PURPOSE: We analyzed the risk of bladder cancer in offspring according to parental and sibling cancer using the national Swedish Family Cancer Database. MATERIALS AND METHODS: Cancer data were obtained from the Swedish Cancer Registry for 1958 to 1996, including 2,105 cases of bladder cancer in offspring. The standardized incidence ratio was used to measure cancer risk in offspring according to familial cancer status. RESULTS: The incidence ratio of bladder cancer increased in Sweden from 1958 to 1996 and it was 3 to 4-fold higher in males than in females. We identified 65 families in which the parents and offspring had bladder cancer with a familial risk of 1.35 (95% confidence interval [CI] 0.97 to 1.79) in sons and 2.29 (95% CI 1.46 to 3.29) in daughters. Discordant cancer sites associated with bladder cancer in the 2 generations were the kidney and thyroid with a standardized incidence ratio of 1.58 (95% CI 1.18 to 2.05) and 1.89 (95% CI 1.00 to 3.05), respectively. Sibling risk was higher compared with offspring risk with a standardized incidence ratio of 2.96 (95% CI 1.41 to 5.08) and in males there was a statistically significant ratio of sibling-to-offspring risk of 2.66 (95% CI 1.29 to 5.45). Patient age at onset modified the familial risk. The highest familial risk of 7.26 (95% CI 2.61 to 14.24) was observed in the brothers of bladder cancer probands diagnosed before age 45 years. CONCLUSIONS: The relatively high ratio of sibling-to-offspring risk as well as observed gender specific effects in bladder cancer may reflect an X linked susceptibility gene.

The nation-wide Swedish family-cancer database--updated structure and familial rates.

The Swedish Family-Cancer Database was expanded to include all Swedes born in 1932 and later (offspring) with their parents, totaling 10.2 million individuals. Cancer cases were retrieved from the Swedish Cancer Registry from the years 1958 to 1998, including over 1 million primary cancers and in situ tumors. Some 10%, of offspring diagnosed with cancer lack any parental information. Incidence rates of cancers were similar in the database and in the Cancer Registry to age 70, but at higher ages the rates in the Database were lower, probably because of selection. The familial risk for all types of cancer in offspring was 1.73 when a parent had the same type of cancer. The familial rates were increased for all main cancer sites, except for the upper aerodigestive tract, stomach, liver, pancreas and bone marrow (leukemia). The rates were 7.47 for thyroid, 4.69 for testis, and over 2.00 for melanoma, ovary, prostate, skin, endocrine glands and endometrium.

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.

Specific DNA adducts induced by some mono-substituted epoxides in vitro and in vivo.

Alkyl epoxides are important intermediates in the chemical industry. They are also formed in vivo during the detoxification of alkenes. Alkyl epoxides have shown genotoxicity in many toxicology assays which has been associated with their covalent binding to DNA. Here aspects of the formation and properties of DNA adducts, induced by some industrially important alkenes and mono-substituted epoxides are discussed. These include propylene oxide, epichlorohydrin, allyl glycidyl ether and the epoxy metabolites of styrene and butadiene. The major DNA adducts formed by epoxides are 7-substituted guanines, 1- and 3-substituted adenines and 3-substituted cytosines. In addition, styrene oxide and butadiene monoepoxide are able to modify exocyclic sites in the DNA bases, the sites being in the case of styrene oxide N(2)- and O(6)-positions of guanine, N(6)-adenine as well as N(4)-and O(2)-cytosine. In vivo the main adduct is the 7-substituted guanines. The 1-substituted adenines have also shown marked levels, and these adducts should also be targets in biomonitoring of human exposures. Due to its low mutagenicity, 7-substituted guanines are considered as a surrogate marker for other mutagenic lesions, e.g. those of 1-adenine or 3-uracil adducts.

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.

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.

Dosimetry of glycidyl ethers in mice by quantification of haemoglobin adducts.

Glycidyl ethers are used in epoxy resins. Epoxy resins are widely used in adhesives and coatings, as well as in electronics and structural composites, thus there is a potential of human exposure to glycidyl ethers. The aim of the present investigation was to explore the utility of haemoglobin adducts for biomonitoring of these types of compounds. Adducts to N-terminal valine were analysed by a modified Edman method with gas chromatographymass spectrometry (or tandem mass spectrometry) for adduct detection and quantification. Groups of three male mice (C3H/Hej) were administered 4 mg/mouse of allyl, butyl, phenyl or cresyl glycidyl ether (AGE, BGE, PGE or CGE) by i.p. injection. Blood samples were collected 24 h after treatment and assayed for haemoglobin adducts using the N-alkyl Edman method. Additional groups of AGE-treated mice were used to study dose response and adduct stability. The experiments with AGE indicate a linear dose response for adduct formation in the dose range studied (0, 2 and 4 mg/mouse). As expected for stable haemoglobin adducts, about 50% of the initial adduct level remained 21 days after exposure.

Adduct formation on DNA and haemoglobin in mice intraperitoneally administered with styrene.

Styrene-specific N-7- and O6-guanine DNA adducts and N-terminal valine adducts were determined in mice tissues after the intraperitoneal administration of styrene. Blood, liver, lungs and spleen were collected 3 h after administration of various doses (from 0 to 4.35 mmol/kg b.w.) of styrene. DNA adducts were analysed by the modified 32P-postlabelling assay and N-terminal valine adducts were detected by GC-MS according to the modified Edman degradation technique. In the dose-range studied, for all adducts a clear dose-response relationship was observed. 7-Alkylguanines and O6-styrene guanine adducts were most abundant in lungs, approximately 30% more than in liver and spleen. In all analysed tissues 7-alkylguanines were more abundant than O6-styrene guanine adducts. We found a considerably lower rate of N-terminal valine adduct formation as compared with both DNA adducts. The ratio between 7-alkylguanines and O6-guanine adducts was 1.9, 1.6 and 7.8 in liver, lung and spleen, respectively. In vitro determination of both DNA adducts by 32P-postlabelling resulted also in a lower ratio than that reported earlier using an HPLC analysis. All correlation's between dose, haemoglobin and DNA adducts were very high and significant. However, at the highest injected doses the adduct formation showed a levelling off. To explain this phenomenon a model simulation was performed revealing that 3 h after the injection of the higher doses styrene was not completely converted into styrene-7,8-oxide.

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.

7-Alkylguanine adducts of styrene oxide determined by 32P-postlabeling in DNA and human embryonal lung fibroblasts (HEL).

The modified 32P-postlabeling method was used for the detection of N-7-(2-hydroxy-phenylethyl) guanine adducts in DNA and human embryonal lung (HEL) cells treated in vitro with styrene oxide (SO). The total recovery of 7-alkylguanine adducts of styrene oxide in DNA analysed by 32P-postlabeling assay was 4.1 +/- 0.6%. The disappearance of 7-alkyldeoxyguanosine monophosphate adducts from SO-modified DNA at 37 degrees C showed a half-life of 19 h. The levels of 7-alkylguanine DNA adducts and single-strand breaks (SSBs) in DNA were determined in HEL cells treated with SO for 3 and 18 h. In the 3 h treatment there was a concentration-dependent increase of both 7-alkylguanine adducts and SSBs in DNA (r = 0.98, P = 0.012 and r = 0.99, P = 0.003, respectively). We found a significant correlation between 7-alkylguanine DNA adducts and SSBs in DNA (r = 0.98, P = 0.011). In HEL cells approximately 3-fold higher levels of both 7-alkylguanine DNA adducts and SSB in DNA were found after the SO (100 microM) treatment for 3 h than after the treatment for 18 h. A significant concentration-dependent increase was found only for SSB ( r = 0.95, P = 0.024) in the 18 h treatment with SO. There was no significant correlation between 7-alkylguanine adducts and SSBs (r = 0.72, P = 0.15). Our data suggest relatively fast removal of both 7-alkylguanine adducts of SO and DNA SSBs.