Biomedical subjects

J A Swenberg

Publications and source records attributed to J A Swenberg.

At least 55 records · Page 3Linked to original sources

PubMed1999

Formation and repair of DNA adducts in vinyl chloride- and vinyl fluoride-induced carcinogenesis.

Vinyl chloride is a known human and animal carcinogen that induces angiosarcomas of the liver. We review here studies on the formation and repair of DNA adducts associated with vinyl chloride and vinyl fluoride in exposed and control rodents and unexposed humans. These vinyl halides induce etheno (epsilon) adducts that are identical to those formed after lipid peroxidation. Of these adducts, N2,3-ethenoguanine (epsilon G) is present in greatest amounts in tissues of exposed animals. After exposure to vinyl chloride for four weeks, epsilon G levels attain steady-state concentrations, such that the amount of newly formed adducts equals the number of adducts that are lost each day. We report the first dosimetry of epsilon G in rats exposed to 0, 10, 100 or 1100 ppm vinyl chloride for five days or four weeks. The number of adducts increased in a supralinear manner. Exposure to 10 ppm vinyl chloride for five days caused a two- to threefold increase in epsilon G over that of the controls, while four weeks' exposure resulted in a fivefold increase. This was confirmed with [13C2]vinyl chloride and by measuring exogenous and endogenous adducts in the same animals. Exposure to 100 ppm vinyl chloride for four weeks caused a 25-fold increase in epsilon G levels over that found in control rats, while exposure to 1100 ppm resulted in a 42-fold increase. The amount of endogenous epsilon G was similar in liver DNA from rats and humans. A comparable response to exposure was seen in rats and mice exposed to 0, 25, 250 or 2500 ppm vinyl fluoride for 12 months. There was a very high correlation between epsilon G levels in rat and mouse liver at 12 months and the incidence of haemangiosarcoma at two years. We were able to demonstrate that the target cell population for angiosarcoma, the nonparenchymal cells, contained more epsilon G than hepatocytes, even though nonparenchymal cells are exposed by diffusion of vinyl halide metabolites formed in hepatocytes. The expression of N-methylpurine-DNA glycosylase mRNA was induced in rat liver after exposure to either 25 or 2500 ppm vinyl fluoride. When this induction was investigated in hepatocytes and nonparenchymal cells, it was found that the latter had only 20% of the N-methylpurine-DNA glycosylase mRNA of hepatocytes, and that only the hepatocytes had induction of this expression after exposure to vinyl fluoride. Thus, the target cells for vinyl halide carcinogenesis have much lower expression of this DNA repair enzyme, which has been associated with etheno adduct repair.

Animals↗

PubMed1998

Gentle in situ liver manipulation during organ harvest decreases survival after rat liver transplantation: role of Kupffer cells.

BACKGROUND: The etiology of primary graft nonfunction and dysfunction is unknown but most likely involves Kupffer cell-dependent reperfusion injury. However, the donor operation and surgical technique may also have an effect on the outcome after transplantation. Because liver manipulation during harvest cannot be prevented completely with standard procedures, its effect on survival was assessed here. METHODS: Donor livers were harvested from female Sprague-Dawley rats (200-230 g). Briefly, after minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 subsequent minutes. At 25 min, all livers were perfused with cold University of Wisconsin solution via the portal vein, and transplantation was performed after cold storage (1 hr). In some rats, Kupffer cells were destroyed with gadolinium chloride or inactivated with dietary glycine before harvest. Survival, proteolytic activity in the rinse effluent, serum transaminases, trypan blue distribution to index microcirculation, and histology were compared. RESULTS: In the nonmanipulated group, survival was 100% after transplantation; however, gentle manipulation decreased survival by 70%. Further, manipulation elevated transaminases fivefold and caused about 200% necrosis. At harvest, proteolytic activity and the time for trypan blue to distribute homogeneously were elevated three- to eightfold by manipulation. Gadolinium chloride and glycine prevented the effects of manipulation on all parameters studied. CONCLUSION: These data indicate for the first time that brief, gentle manipulation of the donor liver has a marked detrimental effect on survival by priming or activating Kupffer cells. This may represent an important early event in pathogenesis, because Kupffer cells play an important role in primary graft nonfunction.

Adenosine↗

PubMed1998

Highly sensitive apurinic/apyrimidinic site assay can detect spontaneous and chemically induced depurination under physiological conditions.

One of the most prevalent lesions in DNA is the apurinic/apyrimidinic (AP) site, which is derived from the cleavage of the N-glycosyl bond by DNA glycosylase or by spontaneous depurination. AP sites are repaired by AP endonucleases during the process of base excision repair; however, an imbalance in this DNA repair system may cause mutations as well as cell death. We have established a sensitive and convenient slot-blot method to detect AP sites in genomic DNA using a novel aldehyde reactive probe (ARP), which reacts with the aldehydic group of ring-opened AP sites. The reaction of 1 mM of ARP with 15 microg of genomic DNA containing AP sites at 37 degrees C was completed within 1 min. The AP site-ARP complex was remarkably stable during incubation in TE buffer, even at 100 degrees C for 60 min. The sensitivity of this assay enables detection of 2.4 AP sites per 10(7) bases. By using this ARP-slot-blot assay, the rate of spontaneous depurination of calf thymus DNA was determined. Under physiological conditions, AP sites were increased at 1.54 AP sites/10(6) nucleotides/day (9000 AP sites/cell/day). This highly sensitive assay allows us to determine the endogenous level of AP sites in genomic DNA, as well as to investigate whether DNA-damaging agents cause imbalances of base excision/AP endonuclease repair in vivo and in vitro.

B-Lymphocytes↗

PubMed1998

Quantitative analysis of 1,3-butadiene-induced DNA adducts in vivo and in vitro using liquid chromatography electrospray ionization tandem mass spectrometry.

1,3-Butadiene (BD) is a high volume industrial chemical which is known as a multi-site rodent carcinogen and is classified as a probable human carcinogen. Covalent interactions of the reactive epoxy metabolites of BD with DNA lead to the formation of DNA adducts which may cause mutations and tumor formation. In the present work, liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was employed for analyses of BD-induced DNA adducts in vitro and in vivo. Selected reaction monitoring (SRM) using the fragmentation of the [M + H]+ ions of the adducts to the corresponding protonated nucleobases under collision-induced dissociation was performed. Quantitation was based on isotope dilution with 13C- and 15N-labeled internal standards. The methods were applied in vitro [calf thymus DNA and TK6 cell cultures treated with epoxy metabolites of BD, 3,4-epoxy-1-butene (EB) and diepoxybutane (DEB)] and in vivo [DNA isolated from tissues of BD-exposed laboratory animals]. Two regioisomers of N-7-EB-guanine adducts, N-7-(2-hydroxy-3-buten-1-yl)guanine (N-7-EB-Gua I) and N-7-(1-hydroxy-3-buten-2-yl)guanine (N-7-EB-Gua II) and two N-3-EB-adenine isomers, N-3-(2-hydroxy-3-buten-1-yl)adenine and N-3-(1-hydroxy-3-buten-2-yl)adenine (N-3-EB-Ade I and II), were found in EB-exposed samples. N-7-(2',3',4'-trihydroxybut-1'-yl)guanine (N-7-THB-Gua), N6-(2',3',4'-trihydroxybut-1'-yl)adenine (N6-THB-Ade), and N-3-(2',3',4'-trihydroxybut-1'-yl)adenine (N-3-THB-Ade) were detected in DEB-treated DNA. DNA isolated from liver and lung of rats and mice exposed to 1250 ppm BD for 2 weeks contained both regioisomers of N-7-EB-Gua and N-3-EB-Ade, as well as N-7-THB-Gua and N6-THB-Ade. The methods developed in this work provide the means to study accumulation, repair and dose-response relationships of BD-DNA adducts in vivo. Although less sensitive than gas chromatography/electron capture negative ionization high-resolution mass spectrometry (GC/ECNI-HRMS), LC/ESI(+)-MS/MS in the SRM mode is extremely useful for analysis of BD-DNA adducts, which are not amenable to GC and derivatization owing to the presence of several adjacent polar functional groups. Using LC/ESI-MS/MS and isotope dilution, multiple structurally diverse BD-DNA adducts can be analyzed simultaneously in the same sample with minimal sample preparation.

Animals↗

PubMed1998

Application of gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry for analysis of DNA and protein adducts.

The analytical potential of gas chromatography/electron capture negative chemical ionization high-resolution mass spectrometry (HRMS) for characterization and quantitation of DNA and hemoglobin adducts was demonstrated using three model compounds: N2, 3-ethenoguanine (EG), 7-(2-hydroxyethyl)guanine (7-HEG), and N-(2-hydroxyethyl)valine (HEV). At a resolving power of 10 000, the signal-to-noise (S/N) ratios obtained from quantitative selected ion monitoring (SIM) experiments using biological samples were comparable to or better than existing unit mass resolution experiments due to the reduction of chemical noise from the use of narrower mass windows. The specificity gained by HRMS was essential for quantitation of ultratrace amounts near the limit of detection since coeluting interferences of the analyte or internal standard can lead to inaccurate measurement of response factors. The limit of detection (LOD) was 100 amol (S/N = 5) using a pure standard of TTB2-EG. The LOD for complete assays using spiked samples was 500 amol (S/N = 5) for EG and 600 amol (S/N = 5) injected for 7-HEG. The standard deviation (SD) for the HRMS quantitative measurements was typically less than 10%. The SD for the complete biological assays as determined by spiking replicate samples was less than 15%. This method has adequate sensitivity and specificity to accurately measure DNA and protein adducts as low as endogenous concentrations in rodent and human tissues.

Animals↗

PubMed1998

Quantitation of 1,N6-ethenoadenine in rat urine by immunoaffinity extraction combined with liquid chromatography/electrospray ionization mass spectrometry.

A fast, highly specific analytical method was developed to quantify 1,N6-ethenoadenine (epsilonA) in urine of rats. epsilonA is a highly mutagenic DNA adduct generated by vinyl chloride (VC) exposures as well as endogenously from lipid peroxidation. epsilonA was concentrated through extraction from rat urine by immunoaffinity chromatography and quantitated by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The average epsilonA recovery by immunoaffinity extraction was 66%. The LC/ESI-MS selected-ion monitoring (SIM) of the response ratio of epsilonA to its isotopically labeled internal standard [15N5]epsilonA was linear (r2 = 0.999) and reproducible from 0.15 to 30 pmol/injection. The detection limit obtained in the routine analysis of urine of unexposed rats was 270 fmol/sample with a signal-to-noise ratio (S/N) 3:1. The concentration of endogenous epsilonA was determined to be 21.6 +/- 14.8 pmol/mL (3 rats). Following portal injection of chloroethylene oxide (CEO; the putative active metabolite of VC), the rate of epsilonA excretion in urine was greatest from 0 to 24 h, with approximately 90% of the CEO-induced epsilonA excreted. By 132 h, the excretion of epsilonA was similar to pretreatment amounts. The accuracy of the quantitation was 107 +/- 6% (n = 4), established by analyzing urine of an unexposed rat spiked with authentic epsilonA. These data indicate that the LC/ESI-MS with immunoaffinity extraction method is precise and accurate for epsilonA quantification. The measurement of epsilonA in urine provides a potential biomarker for exposure to chemicals and processes that form this adduct.

Adenine↗

PubMed1998

Detection of 1,N6-ethenoadenine in rat urine after chloroethylene oxide exposure.

The four etheno adducts of vinyl chloride formed in DNA, 1,N6-ethenoadenine (epsilonA), 3,N4-ethenocytosine, 1,N2-ethenoguanine and N2,3-ethenoguanine were previously reported to be released from DNA by a family of enzymes in the base-excision repair pathway (Dosanjh et al., Proc. Natl Acad. Sci. USA, 91, 1024-1028, 1994; Hang et al., Carcinogenesis, 17, 155-157, 1996; Hang et al., Proc. Natl Acad. Sci. USA, 94, 12869-12874, 1997). Adducts excised from DNA by glycosylases are usually excreted in urine and have been reported to be potential biomarkers of DNA damage in exposed individuals. In this study, we report the detection of epsilonA in the urine of rats exposed to chloroethylene oxide (CEO) using immunoaffinity columns made with specific monoclonal antibodies for enrichment, followed by quantitation by HPLC with fluorescence detection. Chemical analysis of urine samples revealed the presence of a compound chromatographically identical to authentic epsilonA standard. This compound was confirmed by mass spectral analysis. EpsilonA was present in urine of control and CEO-treated rats, with the latter having up to 50-fold greater amounts. The cumulative excretion of epsilonA reached a plateau between 24 and 48 h post-exposure. While it is clear that CEO treatment results in increased excretion of epsilonA, the exact source of the adduct is unknown. When rats were administered epsilonA i.v., approximately 10% of the administered dose was excreted in urine. This research demonstrates that urinary excretion of epsilonA may be a potential biomarker for in vivo alkylation of DNA and nucleotide pools.

Adenine↗

PubMed1998

Molecular dosimetry of DNA adducts in the medaka small fish model.

Small fish models are being used with increasing frequency for carcinogenicity testing and comparative cancer research in the US, Canada and Europe. However, there is a need to further define the early biochemical events of carcinogenesis in these species. Identification and quantitation of DNA adducts can integrate all of the various factors involved in chemical exposure, uptake, distribution and biotransformation of a putative carcinogen. In the present study, Japanese medaka (Oryzias latipes) were exposed to the alkylating agent, diethylnitrosamine (DEN), in the ambient water. Liver DNA was analyzed for O6-ethylguanine (O6EG), O4-ethylthymidine (O4ET) and O2-ethylthymidine (O2ET) by the immuno-slot-blot technique, using monoclonal antibodies against each adduct of interest. While fish exposed to 10 p.p.m. DEN had liver DNA adduct concentrations at or only slightly higher than background levels, those exposed to 100 p.p.m. DEN averaged 34 and 53 pmol O6EG/micromol guanine, 15 and 41 pmol O2ET/micromol thymidine and 2 and 6 pmol O4ET/micromol thymidine at 0 and 24 h post-exposure, respectively. The results of this study show that, under these short-term exposure conditions, ethyl-DNA adducts appear to accumulate in medaka liver tissue in a sublinear (i.e. non-linear) fashion after aqueous exposure to DEN. Thus, critical DNA repair enzymes such as O6-alkylguanine DNA alkyltransferase, which are relatively efficient at lower carcinogen levels, are probably saturated at the 100 p.p.m. concentration level of DEN.

Animals↗

PubMed1997

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.

Alkylation↗

PubMed1997

Synthesis, characterization, and in vitro quantitation of N-7-guanine adducts of diepoxybutane.

Diepoxybutane (DEB) is an important metabolite of 1,3-butadiene (BD), a high-volume industrial chemical classified as a probable human carcinogen. Rodent inhalation studies show strikingly high sensitivity of mice to carcinogenic effects of butadiene compared to rats, which has been linked to differences in metabolism. Both species convert BD to 3,4-epoxy-1-butene (EB), but mice further oxidize a significantly greater part of EB to DEB. DEB is a potent bifunctional genotoxic agent which is 100-fold more mutagenic than EB and is likely to be involved in BD-induced carcinogenesis. Identification of specific BD-induced DNA adducts is critical to understanding the mechanism of its biological activity. We have previously described reactions of EB with guanine and adenine as nucleobases, nucleosides, and constituents of DNA. In this work, DEB-induced guanine adducts were isolated and structurally characterized by UV spectroscopy, mass spectrometry, and nuclear magnetic resonance. When guanosine was reacted with DEB in glacial acetic acid followed by hydrolysis in hydrochloric acid, three products were isolated: N-7-(2',3',4'-trihydroxybut-1'-yl)guanine (DEB-Gua I, major adduct), N-7-(2',4'-dihydroxy-3'-chlorobut-1'-yl)guanine (DEB-Gua II), and N-7-(2',3'-dihydroxy-4'-acetoxybut-1'-yl)guanine (DEB-Gua III). We suggest initial formation of the N-7-(2'-hydroxy-3',4'-epoxybut-1'-yl)guanine intermediate followed by nucleophilic substitution at the 3',4'-epoxy ring with hydroxide, chloride, or acetate anions to give DEB-Gua I, II, or III, respectively. DEB-Gua I and the epoxy intermediate were also isolated from hydrolysates of DEB-exposed calf thymus DNA (CT DNA). N-7-Guanine adducts are known to undergo spontaneous and enzymatic depurination producing apurinic sites. If not repaired before DNA replication, apurinic sites can give rise to mutations and ultimately cancer. The extent of alkylation at the N-7 of guanine in DEB-exposed DNA (58.7 +/- 1.1 adducts/10(3) normal guanines) was similar to that previously reported for CT DNA exposed to EB at the same molar ratio. Since EB and DEB appear to induce comparable levels of overall DNA alkylation at the conditions applied in this work, other factors, such as formation of DNA cross-links by DEB but not EB or differences in repair of EB and DEB adducts, may be responsible for the differences in mutagenicity.

Carcinogens↗

PubMed1997

Adenine adducts with diepoxybutane: isolation and analysis in exposed calf thymus DNA.

1,3-Butadiene (BD) is a high-volume industrial chemical and a common environmental pollutant. Although BD is classified as a "probable human carcinogen", only limited evidence is available for its tumorigenic effects in occupationally exposed populations. Animal studies show a surprisingly high sensitivity of mice to the carcinogenic effects of BD compared to rats (approximately 10(3)-fold), making interspecies extrapolations difficult. Identification and quantitation of specific BD-induced DNA adducts are important for improving our understanding of the mechanisms of BD biological effects and for explaining the observed species differences. Covalent binding of BD to DNA is probably due to its two epoxy metabolites: 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepoxybutane (DEB). Both EB and DEB are direct mutagens producing frameshift and point mutations at both A:T and G:C base pairs. DEB is 100 times more mutagenic than EB and is found in quantity only in tissues of the most sensitive species (mouse). This has led to the suggestion that the higher sensitivity of mice to BD could be due to greater exposure to DEB. The present work was initiated in order to isolate and structurally characterize DEB-induced adenine adducts. The adducts were formed by reacting DEB with free adenine (Ade), 2'-deoxyadenosine (2'-dAdo), and calf thymus DNA followed by HPLC separation and analysis of the products by UV spectrophotometry, electrospray ionization mass spectrometry, and nuclear magnetic resonance. The adenine reaction resulted in three products which were identified as N-3-, N-7-, and N-9-(2'-hydroxy-3',4'-epoxybut-1'-yl)adenine. These adducts underwent acid-catalyzed hydrolysis to their corresponding (2',3',4'-trihydroxybut-1'-yl)adenines upon heating or storage. The 2'-dAdo reaction with DEB followed by acid hydrolysis yielded a single adduct, N6-(2',3',4'-trihydroxybut-1'-yl)adenine (N6-DEB-Ade). N-3-DEB-Ade and N6-DEB-Ade were also found in hydrolysates of calf thymus DNA exposed to DEB. The amounts of N-3-DEB-Ade (13/10(3) normal Ade) and N6-DEB-Ade (5/10(3) normal Ade) were slightly lower than those of the corresponding EB-induced adducts in similar experiments, suggesting comparable reactivity of the two epoxy metabolites of BD toward adenine in DNA. The findings of this study provide a basis for future analyses of BD-induced adenyl DNA adducts in vitro and in vivo.

Adenine↗

PubMed1997

Identification and quantitation of DNA adducts from calf thymus DNA exposed to 3,4-epoxy-1-butene.

3,4-Epoxy-1-butene (EB) is the major mutagenic metabolite of butadiene (BD), an important industrial chemical classified as a probable human carcinogen. Although the mechanism of carcinogenicity of EB is not known, its reactions with nucleophilic sites of DNA giving pro-mutagenic lesions are likely to constitute the early crucial step in multistage carcinogenesis. This study was conducted to characterize the adducts formed from reactions of EB with the most nucleophilic DNA nucleobases, adenine (Ade) and guanine (Gua), as free nucleobases, 2'-deoxyribonucleosides and constituents of calf thymus DNA (CT DNA) in order to provide insight into the nature of DNA modification by EB. The adducts were isolated using HPLC separation coupled with diode array detection (DAD) and structurally characterized from their electronic, mass- and nuclear magnetic resonance spectra. Four EB-adenine products were identified as N-1-(2-hydroxy-3-buten-1-yl) adenine (EB-Ade I), N-1-(1-hydroxy-3-buten-2-yl) adenine (EB-Ade II), N-3-(2-hydroxy-3-buten-1-yl) adenine (EB-Ade III) and N-3-(1-hydroxy-3-buten-2-yl) adenine (EB-Ade IV). Two previously reported guanine adducts: N-7-(2-hydroxy-3-buten-1-yl) guanine (EB-Gua I) and N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-Gua II) were also collected. The purified adducts were used as reference compounds to detect and quantitate the corresponding adduct species formed in calf thymus DNA incubated with EB. All six adducts were detected in treated DNA. The N-7 position of guanine was the most reactive in DNA followed by N-3 of adenine and N-1 of adenine. The formation of N-1 and N-3-adenine adducts (EB-Ade I, 1.2 +/- 0.36; EB-Ade II, 0.8 +/- 0.27; EB-Ade III, 2.7 +/- 0.38; EB-Ade IV, 5.9 +/- 0.68 nmol/micromol Ade) in CT DNA was approximately one-tenth that of EB-guanine adducts (50.7 +/- 2.37 and 47.9 +/- 3.6 nmol/micromol Gua, respectively). The N-1-EB-Ade adducts detected in this study are likely to be the precursors of previously reported N6-EB-adenine adducts (Koivisto et al., 1995) through Dimroth rearrangement. Since BD and EB induce significant numbers of point mutations at A:T base pairs, the EB-adenine adducts may represent important lesions involved in BD-induced mutagenesis and carcinogenesis.

Adenine↗

PubMed1997

Mutagenicity of vinyl chloride and its reactive metabolites, chloroethylene oxide and chloroacetaldehyde, in a metabolically competent human B-lymphoblastoid line.

Vinyl chloride (VC), a known human and rodent carcinogen, is metabolically activated by cytochrome P450 to chloroethylene oxide (CEO), which can rearrange to chloroacetaldehyde (CAA) or undergo hydrolysis. To further understand the roles of CEO and CAA in VC mutagenesis, the types and frequencies of mutations induced at the hypoxanthine (guanine) phosphoribosyl-transferase (hprt) locus were examined in a human B-lymphoblastoid line constitutively expressing human cytochrome P450 2E1 (H2E1 cells). VC was toxic and mutagenic to H2E1 cells as a function of incubation time; exposure to 7.5% VC in air resulted in 75% survival and an hprt mutant frequency of 42 x 10(-6) after 48 h, compared to 5.7 +/- 2.7 x 10(-6) for unexposed cells. The exposure of H2E1 cells to 0.8 to 15.0% VC in air produced similar mutant frequencies without a clear dose-response relationship, suggesting saturation of metabolic activation. Both CEO and CAA exhibited dose-dependent increases in cell killing and mutant frequency in H2E1 cells. Treatment with 16 microM CEO for 24 h resulted in 75% survival and an induced mutant frequency of 23 x 10(-6), while 16 microM CAA produced 5% survival and an induced mutant frequency of 20 x 10(-6). Structural alterations at the hprt locus in independent thioguanine-resistant clones were examined by Southern blot analysis of Pst I-digested DNA with a full-length human hprt cDNA probe. Ten percent (5/50) of VC-induced and 18% (7/38) of CEO-induced mutants showed detectable deletions, compared with 45% (9/20) of CAA-induced mutants. Thus, VC and CEO displayed similar toxicity/mutation profiles and a similar frequency of large deletions, whereas CAA displayed greater toxicity and a larger frequency of deletion mutations. These results suggest that the majority of mutations induced by VC occur through its metabolite, CEO.

Acetaldehyde↗

PubMed1997

A review of the chronic toxicity, carcinogenicity, and possible mechanisms of action of inorganic acid mists in animals.

Occupational exposure to inorganic acid mists containing sulfuric acid has been associated with increased laryngeal cancer. The primary objective of this review was to compile the literature regarding chronic toxicity and carcinogenicity of inorganic acid mists in laboratory animals. Several chronic toxicity studies had exposures of 1 year or longer. Whereas numbers of animals were limited, no evidence of neoplastic or preneoplastic lesions was reported. Two studies evaluated the carcinogenicity of inorganic acid mists in rats; however, one was limited by a short duration of exposure and the other did not achieve a maximum tolerated dose. A large lifetime study in hamsters evaluated the carcinogenicity of 100 mg/M3 sulfuric acid mist, as well as its ability to act as a promoter or co-carcinogen for benzo(a)pyrene. No evidence of carcinogenic potential was shown. Although an increase in papillomas was noted in the benzo(a)pyrene + H2SO4 group, the co-carcinogenic or promoting potential was considered equivocal. Thus, no evidence from experimental animals strongly supports or refutes the induction of cancer by inorganic acid mists. A possible mechanism that could be associated with inorganic acid mist carcinogenicity relates to the genetic consequences of lowering the pH. Reduced pH can induce chromosomal aberrations, enhance depurination, and deamination of cytidine in DNA. This mechanism has not been evaluated in tissues of the respiratory tract.

Aerosols↗

PubMed1997

Clinical relevance of laboratory and animal data on tamoxifen.

Tamoxifen is being evaluated in clinical trials as a preventive agent in women at high risk for breast cancer. This new, potentially long-term therapeutic role has generated some concerns regarding safety, based on the results of animal studies that demonstrated an increased incidence of liver cancer in rats and the formation of DNA adducts. However, direct extrapolation of data from rats to risk estimates in women does not appear to be appropriate. Studies have shown that there are substantial species-related differences in activation and detoxication of tamoxifen that directly affect the formation of DNA adducts. This major species difference in DNA adduct formation appears to be the result of differences in metabolic activation and detoxication; humans form much less of the electrophilic metabolites that bind to DNA and rapidly detoxify those that are formed.

Animals↗

PubMed1996

Macromolecular adducts of butadiene.

Butadiene (BD) is an important industrial chemical classified as a probable human carcinogen. Marked species differences in susceptibility to the carcinogenic effects of BD have been observed, possibly due to the differences in its metabolism. In this work, guanine and adenine adducts formed by the reactive metabolites of BD in vitro were isolated and structurally characterized by UV spectroscopy, liquid secondary ion mass spectrometry and tandem mass spectrometry, electrospray mass spectrometry and nuclear magnetic resonance spectroscopy. The adducts were prepared by reacting purine nucleobases or nucleosides with epoxybutene (EB) or diepoxybutane (DEB) followed by HPLC separation. The reaction of guanine (Gua) with EB resulted in two isomeric products, N7-(2-hydroxy-3-buten-1-yl)guanine (EB-Gua I) and N7-(1-hydroxy-3-buten-2-yl)guanine (EB-Gua II). The reaction of adenine at N3 led to the formation of N3-(2-hydroxy-3-buten-1-yl)adenine (EB-Ade I) and N3-(1-hydroxy-3-buten-2-yl) (EB-Ade II). The major guanine adduct with DEB was identified as N7-(2',3', 4'-trihydroxybutyl)guanine (DEB-Gua-I). Three products formed from the reaction of DEB with adenine at pH 7 were identified as N3, N7 and N9-(2',3',4'-trihydroxybutyl)adenines (DEB-Ade I, II and III, respectively). Our results indicate that nucleophilic nitrogens of guanine and adenine first attack one of the epoxy groups of DEB giving (2'-hydroxy-3',4'-epoxybutane-1-yl) intermediates which can be rapidly hydrolyzed to the corresponding (2',3',4'-trihydroxybutyl) adducts or form cross-links with DNA or proteins. N7 and N3 adducts of Ade and Gua are expected to undergo spontaneous depurination and repair by methylpurine glycosylase and therefore may be useful as biomarkers of exposure in urine. The preliminary data on quantification of EB-induced N-terminal valine hemoglobin adducts in red blood cells of exposed mice and rats using modified Edman degradation followed by GC-NI MS was investigated. The amount of EB-N-terminal valine adducts in mouse globin was about 3 times greater than that in rats which may be explained by higher rates of the formation and/or limited detoxification of EB in mice. Female rats and mice had greater amounts of hemoglobin adducts than males.

Adenine↗

PubMed1996

Correlation of regional and nonlinear formaldehyde-induced nasal cancer with proliferating populations of cells.

Formaldehyde induces nonlinear, concentration-related increases in nasal epithelial cell proliferation and squamous cell carcinomas (SCC) in rats. A formaldehyde carcinogenicity study was conducted in which a major end point was correlation of cell proliferation indices with sites of formaldehyde-induced SCC. A poor correlation in certain sites led to incorporation of the number of cells in each site into the correlation. Rats were exposed (6h/day, 5 days/week) to formaldehyde (0, 0.7, 2, 6, 10 or 15 ppm) for up to 24 months with interim sacrifice time points at 3, 6, 12, and 18 mo. A unit length labeling index (ULLI; S-phase nuclei/mm basement membrane) was determined for specific nasal regions in addition to a population-weighted ULLI (PWULLI). The PWULLI was defined as the product of regional ULLI and total number of nasal epithelial cells in the respective site. Nasal SCC sites of origin were mapped. Formaldehyde induced SCC in a highly nonlinear fashion, with no observed effect at the level of 2 ppm, a minimal response at 6 ppm, and a sharp increase at 10 and 15 ppm. The tumor incidence was 1, 22, and 47% at 6, 10 and 15 ppm, respectively. ULLI was significantly (P<0.05) increased at 10 and 15 ppm but not at the lower concentrations. There was a good correlation between PWULLI and regional tumor incidence (R(2) = 0.88), while the correlation of regional SCC with ULLI was relatively poor (R(2) = 0.46). We conclude that target cell population size and sustained increases of cell proliferation in these populations, determined by differences in regional airflow-driven formaldehyde binding to DNA dose to these sites, coupled with the known nonlinear kinetics of formaldehyde binding to DNA, can together account for the nonlinearity and site specificity of formaldehyde-induced nasal SCC in rats.

Animals↗

PubMed1996

Quantitative analysis of the DNA adduct N2,3-ethenoguanine using liquid chromatography/electrospray ionization mass spectrometry.

The need for specificity and sensitivity in the analysis of DNA adducts has led the development of GC/MS methods. Such methods require chemical derivatization (i.e. silylation, electrophore labelling), which can also bring its own sets of problems, including the production of artifacts, interferences and sample to sample variability in derivatization. To obviate such problems, a liquid chromatographic/electrospray ionization mass spectrometric (LC/ESI-MS) method was developed to quantify N2,3-ethenoguanine (epsilon Gua), a promutagenic DNA adduct of vinyl chloride exposure. The response of epsilon Gua to isotopically labelled internal standard [13C4]epsilon Gua was linear (r2 = 0.999) and reproducible from 0.027 to 0.538 pmol microliter-1. We obtained an accuracy of 86 +/- 14% by analyzing chloroethylene oxide (CEO)-treated calf thymus DNA enriched with authentic epsilon Gua. The analysis of CEO-treated calf thymus DNA samples not enriched with authentic epsilon Gua provided a precision of 15%. The detection limits with a signal-to-noise ratio (S/N) 2.5:1 were obtained in the determination of authentic epsilon Gua at 5 fmol per injection. The detection limit obtained in the routine analysis of the biological samples was 50 fmol epsilon Gua with S/N = 3:1. The applicability of the method was established by determining epsilon Gua in rats treated with CEO by portal vein injection and an unexposed human liver. It was observed that the concentration of epsilon Gua in the rat livers increased with increase in dose and was inversely related to the time after, CEO exposure. This trend suggests rapid repair of the adduct in rat livers. In the human liver DNA sample, epsilon Gua was quantitated at 0.06 +/- 0.01 pmol mg-1 DNA.

Animals↗