Dose responses for the formation of hemoglobin adducts and urinary metabolites in rats and mice exposed by inhalation to low concentrations of 1,3-[2,3-(14)C]-butadiene

Blood and urine were obtained from male Sprague-Dawley rats and B6C3F1 mice exposed to either a single 6 h or multiple daily (5 x 6 h) nose-only doses of 1,3-[2,3- (14)C]-butadiene at atmospheric concentrations of 1, 5 or 20 ppM. Globin was isolated from erythrocytes of exposed animals and analyzed for total radioactivity and also for N-(1,2,3-trihydroxybut-4-yl)-valine adducts. The modified Edman degradation procedure coupled with GC-MS was used for the adduct analysis. Linear relationships were observed between the exposures to 1,3-[2,3-(14)C]-butadiene and the total radioactivity measured in globin and the level of trihydroxybutyl valine adducts in globin. A greater level of radioactivity (ca. 1.3-fold) was found in rat globin compared with mouse globin. When analyzed for specific amino acid adducts, higher levels of trihydroxybutyl valine adducts were found in mouse globin compared with rat globin. Average levels of trihydroxybutyl valine adduct measured in globin from rats and mice exposed for 5 x 6 h at 1, 5 and 20 ppM 1,3-[2,3-(14)C]-butadiene were, respectively, for rats: 80, 179, 512 pM/g globin and for mice: 143, 351, 1100 pM/g globin. The profiles of urinary metabolites for rats and mice exposed at the different concentrations of butadiene were obtained by reverse phase HPLC analysis on urine collected 24 h after the start of exposure and were compared with results of a previous similar study carried out for 6 h at 200 ppM butadiene. Whilst there were qualitative and quantitative differences between the profiles for rats and mice, the major metabolites detected in both cases were those representing products of epoxide hydrolase mediated hydrolysis and glutathione (GSH) conjugation of the metabolically formed 1,2-epoxy-3-butene. These were 4-(N-acetyl-l-cysteine-S-yl)-1,2-dihydroxy butane and (R)-2-(N-acetyl-l-cystein-S-yl)-1-hydroxybut-3-ene, 1-(N-acetyl-l-cystein-S-yl)-2-(S)-hydroxybut-3-ene, 1-(N-acetyl-l-cystein-S-yl)-2-(R)-hydroxybut-3-ene, (S)-2-(N-acetyl-l-cystein-S-yl)-1-hydroxybut-3-ene, respectively. The former pathway showed a greater predominance in the rat. The profiles of metabolites were similar at exposure concentration in the range 1-20 ppM. There were however some subtle differences compared with results of exposure to the higher 200 ppM concentrations. Overall the results provide the basis for cross species comparison of low exposures in the range of occupational exposures, with the wealth of data available from high exposure studies.     

DNA adducts in rats and mice following exposure to [4-14C]-1,2-epoxy-3-butene and to [2,3-14C]-1,3-butadiene

1,3-Butadiene (BD) is a major industrial chemical and a rodent carcinogen, with mice being much more susceptible than rats. Oxidative metabolism of BD, leading to the DNA-reactive epoxides 1,2-epoxy-3-butene (BMO), 1,2-epoxy-3,4-butanediol (EBD) and 1,2:3,4-diepoxybutane (DEB), is greater in mice than rats. In the present study the DNA adduct profiles in liver and lungs of rats and mice were determined following exposure to BMO and to BD since these profiles may provide qualitative and quantitative information on the DNA-reactive metabolites in target tissues. Adducts detected in vivo were identified by comparison with the products formed from the reaction of the individual epoxides with 2'-deoxyguanosine (dG). In rats and mice exposed to [4-14C]-BMO (1-50 mg/kg, i.p.), DNA adduct profiles were similar in liver and lung with N7-(2-hydroxy-3-butenyl)guanine (G1) and N7-(1-(hydroxymethyl)-2-propenyl)guanine (G2) as major adducts and N7-2,3,4-trihydroxybutylguanine (G4) as minor adduct. In rats and mice exposed to 200 ppm [2,3-14C]-BD by nose-only inhalation for 6 h, G4 was the major adduct in liver, lung and testes while G1 and G2 were only minor adducts. Another N7-trihydroxybutylguanine adduct (G3), which could not unambiguously be identified but is either another isomer of N7-2,3,4-trihydroxybutylguanine or, more likely, N7-(1-hydroxymethyl-2,3-dihydroxypropyl)guanine, was present at low concentrations in liver and lung DNA of mice, but absent in rats. The evidence indicates that the major DNA adduct formed in liver, lung and testes following in vivo exposure to BD is G4, which is formed from EBD, and not from DEB.     

Metabolic distribution of radioactivity in Sprague-Dawley rats and B6C3F1 mice exposed to 1,3-[2,3-14C]-butadiene by whole body exposure

The uptake of 1,3-[2,3-(14)C]-butadiene and its disposition, measured as radioactivity in urine, faeces, exhaled volatiles and CO(2) during and following 6 h whole body exposure to 20 ppm butadiene has been investigated in male Sprague-Dawley rats and B6C3F1 mice. Whilst there were similarities between the two species, the uptake and metabolic distribution of butadiene were somewhat different for rats and mice. The major differences observed were in the urinary excretion of radioactivity and in the exhalation of 14C-CO(2). After 42 h from the start of exposure, 51.1% of radioactivity was eliminated in rat urine compared with 39.5% for mouse urine. 34.9% of the recovered radioactivity was exhaled by rats as 14C-CO(2), compared with 48.7% by mice. Excretion of radioactivity in faeces was similar for both species (3.8% for rats and 3.4% for mice). The tissue concentrations of 14C-butadiene equivalents measured in liver, testes, lung and blood of exposed mice were 0.493, 0460, 0.457, and 1.626 nmol/g tissue, respectively. The values for the corresponding rat tissues were 0.869, 0.329, 0.457, and 1.626 nmol butadiene equivalents/g tissue, respectively. For rats, 6.2% of recovered radioactivity (0.288 nmol butadiene equivalents/g tissue) was retained in carcasses whereas for mice the amount was 3.6% (0.334 nmol butadiene equivalents/g tissue). There were also some significant differences between the metabolic conversion of 1,3-[2,3-(14)C]-butadiene and excretion by mice following the 20 ppm whole body exposure compared to previously reported data for nose-only exposure to 200 ppm butadiene [Richardson et al., Toxicol. Sci. 49 (1999) 186]. The main difference between the high- and low-exposure studies was in the exhalation of 14C-CO(2). At the 200 ppm exposure, 40% of the radioactivity was exhaled as 14C-CO(2) by rats whereas 6% was measured by this route for mice. The proportional conversion of butadiene to CO(2) by mice was significantly greater at the low exposure concentration compared with that reported for the higher concentration. This shift was not observed for rats. The difference between species could be caused by a saturation of metabolism in mice between 20 and 200 ppm for the pathways leading to CO(2). Restraint or error in collection of CO(2) in the 200 ppm study could also be factors.     

Micronuclei, DNA single-strand breaks and DNA-repair activity in mice exposed to 1,3-butadiene by inhalation

We investigated single-strand breaks and endonuclease III-sensitive sites in DNA along with gamma-irradiation-specific DNA-repair activity in hepatocytes and frequencies of micronuclei in polychromatic bone-marrow erythrocytes of male NMRI mice (2 months old, weight 30-35 g) during sub-acute inhalation exposure to 1,3-butadiene (28 days, 500 mg/m3) and up to 28 days after the exposure. Concentrations of 1,3-butadiene in blood, an indicator of internal exposure, moderately increased during the exposure period. The most interesting finding was that gamma-irradiation-specific DNA-repair activity gradually increased during exposure, being significantly higher compared with control levels on days 7 and 28 of exposure (P = 0.005 and 0.035, respectively), reaching a maximum on day 1 after the termination of exposure (P = 0.003) and then returning to control levels. A significant correlation between gamma-irradiation-specific DNA-repair activity and the concentration of 1,3-butadiene in blood (R = 0.866, P = 0.050) supports a possible induction of DNA-repair activity by the exposure to 1,3-butadiene and formation of its metabolites. The initial increase in micronucleus frequency (micronuclei per 1000 cells) in the exposed mice continuously decreased from 20.4 +/- 5.1 (day 3) to 15.1 +/- 3.2 (day 28) within the exposure period, and subsequently from 12.4 +/- 5.1 to 4.6 +/- 1.6 in the period following termination of the 1,3-butadiene exposure, while micronucleus frequencies in control animals were significantly lower (from 1.7 +/- 1.5 to 4.2 +/- 0.8).     

Age-, gender-, and species-dependent mutagenicity in T cells of mice and rats exposed by inhalation to 1,3-butadiene

Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.     

Molecular interaction of [2,3-14C] acrylonitrile with DNA in gastric tissue of rat

Acrylonitrile (VCN) is used extensively in polymer industries, and is known to induce gastric cancer following oral administration, A paucity of information exists regarding the mechanism(s) by which acrylonitrile induces gastric neoplasia. The time course for uptake of radioactivity by gastric tissue and covalent binding of [2,3-¹⁴C] VCN or its metabolites to gastric DNA were determined following a single oral dose of 46.5 mg/kg. The rates of DNA synthesis and repair, as measured by unscheduled DNA synthesis in the gastric tissue of VCN-treated rats, were also studied. Maximum tissue uptake and covalent binding of radioactivity to gastric DNA were observed at 15 minutes following [2,3-¹⁴C] VCN administration. At 6 hours following VCN administration, significant inhibition (37% of control) in gastric replicative DNA synthesis was observed. A rebound followed by an increase (211% of control) in replicative DNA synthesis was observed at 24 hours. A three-fold elevation in unscheduled DNA synthesis was observed at 24 hours following treatment with VCN. These results indicate that VCN or its metabolites irreversibly interact with gastric DNA, causing DNA damage. The results also indicate that the delayed VCN-induced DNA repair, determined as unscheduled DNA synthesis, is inefficient for the removal of the resulting DNA lesions.     

In vitro oxidation of [U-14C] palmitate, [1-14C] and [6-14C] glucose in newborn and adult rats and pigs

Studies have been made on the intensity of oxidation of [U-14C]-palmitate, [1-14C]- and [6-14C]-glucose by slices of the liver and skeletal muscles of new-born, 1-day, 5-day and adult Wistar rats and domestic pigs. It was found that the level of 14CO2 production from these substrates is higher in tissues of rats than in those of pigs. At early stages of ontogenesis, in tissues of both species intensive oxidation of glucose is observed together with oxidation of fatty acids. In the course of ontogenetic development, the intensity of glucose utilization significantly decreases, whereas the level of fatty acid catabolism remains relatively unaffected.     

Oxidation of D-[U-(14)C] glucose and [1,12-(14)C] dodecanedioic acid by pancreatic islets from Goto-Kakizaki rats.

In pancreatic islets from hereditarily diabetic GK rats, [1,12 -(14)C] dodecanedioic acid (5.0 mM) was oxidized at a rate representing about 5 % of that of D-[U - (14)C] glucose (8.3 mM). Dioic acid and hexose failed to exert any significant reciprocal effects on their respective oxidation. The production of (14)CO(2) from [1,12 -(14)C] dodecanedioic acid was proportional to its concentration in the 0.2 - 5.0 mM range. These results were essentially comparable to those obtained in islets from control rats. They extend, therefore, to GK rats the knowledge that dodecanedioic acid acts as a nutrient in pancreatic islet cells.     

Formation of ketone bodies from [14C]palmitate and [14C]glycerol by tissues from ketotic sheep

Labelled ketone bodies were produced readily from [U-(14)C]palmitate, [2-(14)C]palmitate and [1-(14)C]glycerol by sheep rumen-epithelial and liver tissues in vitro. On a tissue-nitrogen basis, both tissues had similar capacities for ketogenesis. Palmitate was a ketogenic substrate in both rumen-epithelial tissue and liver, and more of its (14)C appeared in ketone bodies than in the (14)CO(2) liberated. Glycerol was actively metabolized to ketone bodies, but more readily underwent complete oxidation to carbon dioxide; this complete oxidation was most pronounced in rumen-epithelial tissue from ketotic ewes. These experiments with labelled compounds confirm earlier observations that rumen-epithelial tissue, like liver, actively forms ketone bodies from long-chain fatty acids and show further that normal rumen-epithelial tissue can convert palmitate into ketone bodies as readily as into carbon dioxide. Free glycerol, which is metabolized only by liver tissue in non-ruminants, is also metabolized by rumen epithelium. The rumen epithelium thus has unique metabolic capacity among extrahepatic tissues.     

Metabolism of [1-14C]glyoxylate, [1-14C]-glycollate, [1-14C]glycine and [2-14C]-glycine by homogenates of kidney and liver tissue from hyperoxaluric and control subjects

1. The metabolism of [1-(14)C]glyoxylate to carbon dioxide, glycine, oxalate, serine, formate and glycollate was investigated in hyperoxaluric and control subjects' kidney and liver tissue in vitro. 2. Only glycine and carbon dioxide became significantly labelled with (14)C, and this was less in the hyperoxaluric patients' kidney tissue than in the control tissue. 3. Liver did not show this difference. 4. The metabolism of [1-(14)C]glycollate was also studied in the liver tissue; glyoxylate formation was demonstrated and the formation of (14)CO(2) from this substrate was likewise unimpaired in the hyperoxaluric patients' liver tissue in these experiments. 5. Glycine was not metabolized by human kidney, liver or blood cells under the conditions used. 6. These observations show that glyoxylate metabolism by the kidney is impaired in primary hyperoxaluria.     

Metabolism of ent-kaurenol-[17-14C], ent-kaurenal-[17-14C] and ent-kaurenoic acid-[17-14C] by germinating Hordeum distichon grains

Subcellular fractions from germinated barley embryos, chloroplast preparations and whole germinating barley grains are able to carry out the conversions ent-kaurenol → ent-kaurenal → ent-kaurenoic acid → ent-hydroxykaurenoic acid, the initial steps of the biosynthetic pathway to gibberellins. Whole grains, and chloroplasts to a slight extent, incorporate radioactivity from ent-kaurenol-[17-¹⁴C] and ent-kaurenoic acid-[17-¹⁴C] into materials with similar but distinct properties from the gibberellins GA₁, GA₃, GA₄ and GA₇.     

Differences in the conversion of the polyunsaturated fatty acids [1-(14)C]22:4(n-6) and [1-(14)C]22:5(n-3) to [(14)C]22:5(n-6) and [(14)C]22:6(n-3) in isolated rat hepatocytes

The reasons why most cellular lipids preferentially accumulate 22:6(n-3) rather than 22:5(n-6) are poorly understood. In the present work the metabolisms of the precursor fatty acids, [1-(14)C]20:4(n-6), [1-(14)C]22:4(n-6) versus [1-(14)C]20:5(n-3), [1-(14)C]22:5(n-3) in isolated rat hepatocytes were compared. The addition of lactate and L-decanoylcarnitine increased the formation of [(14)C]24 fatty acid intermediates and the final products, [(14)C]22:5(n-6) and [(14)C]22:6(n-3). In the absence of lactate and L-decanoylcarnitine, no [(14)C]24 fatty acids and [(14)C]22:5(n-6) were detected when [1-(14)C]22:4(n-6) was the substrate, whereas small amounts of the added [1-(14)C]22:5(n-3) was converted to [(14)C]22:6(n-3). Lactate reduced the oxidation of [1-(14)C]22:4(n-6) and [1-(14)C]22:5(n-3) while L-decanoylcarnitine did not. No significant differences between the total oxidation or esterification of the two substrates were observed. By fasting and fructose refeeding the amounts of [(14)C]24:4(n-6) and [(14)C]24:5(n-3) were increased by 2.5- and 4-fold, respectively. However, the levels of [(14)C]22:5(n-6) and [(14)C]22:6(n-3) were similar in hepatocytes from fasted and refed versus fed rats. With hepatocytes from rats fed a fat free diet the levels of [(14)C]24 fatty acid intermediates were low while the further conversion of the n-6 and n-3 substrates was high and more equal, approx. 33% of [1-(14)C]22:4(n-6) was converted to [(14)C]22:5(n-6) and 43% of [1-(14)C]22:5(n-3) was converted to [(14)C]22:6(n-3). The moderate differences found in the conversion of [1-(14)C]22:4(n-6) versus [1-(14)C]22:5(n-3) to [(14)C]22:5(n-6) and [(14)C]22:6(n-3), respectively, and the equal rates of oxidation of the two substrates could thus not explain the abundance of 22:6(n-3) versus the near absence of 22:5(n-6) in cellular membranes.     

Absorption and distribution of sodium [2-14C]barbital in tissues of normal and dystrophic mice

The absorption and distribution of [2-14C]barbital after oral administration was studied in various tissues, including skeletal muscle, of normal and dystrophic mice. There appeared to be a more rapid gastric emptying in the mutant homozygote as reflected in lower levels of the drug recuperated from the gastrointestinal tract. This resulted in initially higher plasma and tissue concentrations of barbital in the dystrophic mice. Two hours after oral administration, this kinetic profile was reversed so that less barbital remained in the tissues of the dystrophic mouse. The tissue:plasma concentration ratios were consistently, but not significantly, higher in all tissues of the dystrophic animals. Analysis of the half-life of the drug in both groups suggests that there is an increase in the distribution volume of barbital in the dystrophic mice. The phenomenon of more rapid absorption of the barbiturate seems to be more consistent as the symptoms of the disease progress. The altered absorption and disposition of barbital in various tissues of the dystrophic mouse support the concept that a generalized multisystemic disorder may be crucial to the pathogenesis of murine muscular dystrophy, in contradistinction to a purely myogenic origin.     

Biosynthesis of [14C]zearalenone from [1-14C]acetate by Fusarium roseum 'Gibbosum'.

Addition of [1-14C]acetate or [1,2-14C]acetate to actively growing cultures of Fusarium roseum 'Gibbosum' on rice yielded zearalenone with a specific activity ranging between 1.63 and 46.5 microCi/mmol.     

DNA adduct formation of 14 heterocyclic aromatic amines in mouse tissue after oral administration and characterization of the DNA adduct formed by 2-amino-9H-pyrido[2,3-b]indole (AalphaC), analysed by 32P_HPLC.

Heterocyclic aromatic amines (HAAs) are produced during cooking of proteinaceous food such as meat and fish. Humans eating a normal diet are regularly exposed to these food-borne substances. HAAs have proved to be carcinogenic in animals and to induce early lesions in the development of cancer. DNA adduct levels in mouse liver have been measured by 32P-HPLC after oral administration each of 14 different HAAs. The highest DNA adduct levels were detected for 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-9H-pyrido[2,3-b]indole (AalphaC), respectively. To assess a relative risk in a human population, a relative risk index was calculated by combining the DNA adduct levels in mouse liver with human daily intake of heterocyclic amines in a US and in a Swedish population. Such calculations suggest that AalphaC presents the highest risk for humans, e.g. nine-fold higher compared with the most abundant amines in food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP). Therefore, the distribution of DNA adducts in different tissues of mouse was investigated after oral administration of AalphaC. The highest AalphaC-DNA adduct levels were found in liver (137 adducts/10(8) normal nucleotides) followed by heart, kidney, lung, large intestine, small intestine, stomach and spleen, in descending order. To characterize the chemical structure of the major DNA adduct, chemical synthesis was performed. The major DNA adduct from the in vivo experiments was characterized by five different methods. On the basis of these results, the adduct was characterized as N2-(deoxyguanin-8-yl)-2-amino-9H-pyrido [2,3-b]indole. Considering the abundance of AalphaC not only in grilled meat, but also in other products like grilled chicken, vegetables and cigarette smoke and in light of the results of the present study, it is suggested that the human cancer risk for AalphaC might be underestimated.