Conformation of ring polymers in 2D constrained environments.

The combination of ring closure and spatial constraints has a fundamental effect on the statistics of semiflexible polymers such as DNA. However, studies of the interplay between circularity and constraints are scarce and single-molecule experimental data concerning polymer conformations are missing. By means of atomic force microscopy we probe the conformation of circular DNA molecules in two dimensions and in the concentrated regime (above the overlap concentration c*). Molecules in this regime experience a collapse, and their statistical properties agree very well with those of simulated vesicles under pressure. Some circular molecules also create confining regions in which other molecules are trapped. Thus we show further that spatially confined molecules fold into specific conformations close to those found for linear chains, and strongly dependent on the size of the confining box.

Urinary thiobarbituric acid-reacting substances as potential biomarkers of intrauterine hypoxia.

BACKGROUND: Currently available clinical tools cannot accurately identify the extent of perinatal hypoxic injuries. During hypoxia, reactive oxygen species cause lipid peroxidation of cell membranes, yielding oxidation products that constitute thiobarbituric acid-reacting substances (TBARS). OBJECTIVE: To see if the concentrations of TBARS excreted in urine would be elevated during the first day of life in term and preterm infants following chronic hypoxia or acute asphyxia. DESIGN: Thiobarbituric acid-reacting substances levels were measured by a spectrophotometric assay in urine samples collected from term and near-term (>/= 34 weeks gestation, n = 22), and preterm (<34 weeks gestation, n = 52) infants on the first day of life. PATIENTS: Infants were admitted to the St Peter's University Hospital (New Brunswick, NJ) neonatal intensive care unit from July 1997 to January 1999. Acute asphyxia was defined as umbilical cord blood pH values less than 7.05, or Apgar scores of less than 5 at 5 minutes. Chronic hypoxia was defined as intrauterine growth retardation or low birth weight (small for gestational age) associated with pregnancy-induced hypertension or reversal of umbilical arterial blood flow. RESULTS: Among term infants, urinary TBARS levels were significantly increased following acute asphyxia (P =.02). Levels of TBARS also tended to be elevated following chronic hypoxia. Urinary TBARS levels in term infants tended to be increased in those requiring mechanical ventilation (P =.05) or delivery room resuscitation (P =.15), as well as in those passing intrauterine meconium (P =.13) or having clinical evidence of hypoxic-ischemic encephalopathy (P =.24). CONCLUSIONS: The results show a correlation between elevated urinary TBARS levels in term and near-term infants, and perinatal hypoxia (as determined by low Apgar scores or umbilical cord blood acidosis). We speculate that TBARS concentrations may be useful as a biomarker for perinatal hypoxic injury in newborns. Further studies are needed to determine whether elevations in TBARS levels are better predictors of the extent of hypoxic injury than existing markers.

Application of process chemistry and SAR modelling to the evaluation of health findings of lower olefins.

Epidemiology studies show increased leukemia mortality among styrene butadiene rubber (SBR) workers but not among butadiene monomer production employees. A detailed review of the SBR manufacturing process indicates that sodium dimethyldithiocarbamate (DMDTC) introduced into the SBR manufacturing process for a period in the 1950s coincides with increased leukemia mortality. Using the Computer-Optimized Molecular Parametric Analysis of Chemical Toxicity (COMPACT), we assessed the enzyme (cytochrome P450) substrate specificity of an olefin series including 1,3-butadiene (BD) and also modeled its interaction with DMDTC. These analyses showed correlation of a structural/electronic parameter--the COMPACT radius--with the presence or absence of cytogenetic activity and also found that DMDTC would inhibit the oxidative metabolism of BD at least at high concentrations. Both DMDTC and its diethyl analog (DEDTC) bind with CYP 2E1 and CYP 2A6. Both of these isoforms are important in the initial oxidative metabolism of butadiene and other olefins. In co-exposure studies in mice of DMDTC with BD or with epoxybutene (EB), we found that there was a reduced increase in genotoxic activity based on micronuclei induction compared with BD or EB exposure alone. Treatment with DMDTC significantly increased the protein carbonyl contents of hepatic microsomes compared with that of controls, a finding that may be related to DMDTC's activity as a prooxidant. Co-exposure with DMDTC and EB increased hepatic microsomal carbonyls to levels significantly greater than those of DMDTC-treated mice, while EB administration in the absence of DMDTC did not change protein carbonyls relative to those of controls. The increase in hepatic microsomal protein carbonyls suggests that DMDTC may modulate EB metabolism towards the formation of reactive intermediates that react with proteins. The present molecular modeling and mechanistic studies suggest that co-exposure of BD and DMDTC is a plausible biological hypothesis regarding increased leukemia risk among SBR workers.

DNA-protein crosslink and DNA strand break formation in HL-60 cells treated with trans,trans-muconaldehyde, hydroquinone and their mixtures.

The toxicity of benzene, a human leukemogen and ubiquitous environmental pollutant, is mediated in part by ring-hydroxylated metabolites including hydroquinone (HQ) and ring-opened metabolites including trans,trans-muconaldehyde (muconaldehyde, MUC), and their interactions. DNA-protein crosslinks (DNAPC) and DNA strand breaks (DNASB) are toxic lesions associated with the mechanism(s) of toxicity of carcinogenic compounds. In the present studies, we examined the hypothesis that individual and interactive effects of MUC and HQ are involved in the formation of DNAPC and DNASB. We extended our previous studies on DNAPC induction by MUC in HL-60 cells to HQ and mixtures of MUC and HQ, and determined DNASB levels, including 3'OH DNASB. Treatment of HL-60 cells with 25 to 100 microM HQ followed by incubation for 4 hours resulted in 1.3- to 2.8-fold increases in DNAPC levels compared with control, as determined by a K+/sodium dodecyl sulfate (SDS) precipitation assay. At 25 and 100 microM, MUC was 1.8 and 4.9 fold more effective at inducing DNAPC than HQ. Treatment with equimolar mixtures of 25 or 50 microM MUC and HQ resulted in higher DNAPC formation relative to the DNAPC levels expected if the effects were only additive. 3'OH DNASB levels as determined by the TUNEL assay showed a significant concentration-dependent increase 1 hour after treatment with 5 to 25 microM MUC, whereas HQ treatment had no effect. Cotreatment with 25 and 50 microM MUC/HQ mixtures resulted in significant decreases in TUNEL labeling relative to treatment with MUC alone. HL-60 cells treated with 1 to 50 microM MUC or HQ exhibited concentration- and time-dependent increases in DNASB as determined by the FADU assay, which measures a variety of single- and double-strand breaks and alkali labile sites. Exposure to 10 microM MUC gave QDNASB values (1 QDNASB approximately equals 100 DNASB/cell) of 7.5 +/- 1.2 and 15.4 +/- 1.4 at the 1- and 2-hour time points respectively, compared with 0.1 +/- 3.8 and 0.0 +/- 1.5 for the corresponding time controls. The QDNASB values after treatment with 10 microM HQ were 4.4 +/- 0.7 and 17.7 +/- 2.1 at the 1- and 2-hour time points, respectively, compared with 0.0 +/- 0.5 and 0.0 +/- 1.3 for the corresponding time controls. Induction of DNASB was additive 1 hour after treatment with equimolar MUC/HQ mixtures of 5 to 50 microM. These in vitro findings are significant in that DNAPC and DNASB lesions induced by MUC and HQ as well as their interactions could contribute to benzene-induced hematotoxicity and leukemogenesis.

Micronucleus formation in mouse bone marrow cells in vivo in response to trans, trans-muconaldehyde.

Benzene is a human leukemogen, and a bone marrow toxin and carcinogen in experimental animals. The reactive intermediates involved in benzene toxicity and their mechanism(s) of action have not been clearly delineated. We have investigated the clastogenic and cytotoxic effects of trans,trans-muconaldehyde (MUC), a reactive ring-opened benzene metabolite, in mouse bone marrow cells in vivo. Micronucleus formation was significantly increased when CD-1 mice were treated ip with MUC at 4 and 6 mg/kg/day for two days. These results suggest that leukemogenesis by benzene may be attributable, in part, to MUC-related clastogenic and cytotoxic effects in the bone marrow cells.

Structure-activity relationships in the induction of DNA-protein cross-links by hematotoxic ring-opened benzene metabolites and related compounds in HL60 cells.

Previous studies from our laboratory have demonstrated the formation of DNA-protein cross-links (DNAPC), a potentially cytotoxic and genotoxic lesion induced by many leukemogenic agents, in bone marrow cells of mice administered benzene, however, reactive benzene metabolites involved in DNAPC formation by benzene have not been characterized. The present studies examined DNA PC formation in HL60 cells treated with trans,trans-muconaldehyde (MUC), a hematotoxic ring-opened metabolite of benzene, as well as with MUC metabolites and structurally related compounds. Using a K(+)/SDS precipitation assay for DNAPC determination, concentration- and time-dependent increases in DNAPC formation were observed 2 and 4 h after treatment of HL60 cells with 50, 75 and 100 microM MUC. No increases in DNAPC levels were measured in HL60 cells 4 h after treatment with the MUC metabolites 6-hydroxy-trans,trans-2,4-hexadienal (HO-M-CHO), 6-oxo-trans,trans-2,4-hexadienoic acid (CHO-M-COOH), or trans,trans-muconic acid (HOOC-M-COOH), each at 100 microM. Significant increases in DNAPC levels were observed 4 h after treatment with 500 and 1000 microM HO-M-CHO, but not CHO-M-COOH. No effect on DNAPC levels was observed 4 h after treatment with 100 microM for trans, trans-2,4-hexadienal, trans-2-hexenal, hexanal, trans,trans-2, 4-hexadiene, glutaraldehyde, or acrolein. DNAPC induced by MUC and HO-M-CHO may be cytotoxic lesions, as increases in DNAPC levels by these compounds correlated with decreases in cell viability. Except for acrolein, compounds not inducing DNAPC at 100 microM also did not affect cell viability. These studies suggest that both aldehydic carbons of MUC contribute to DNAPC induction, and that the presence of alpha,beta-unsaturated double bonds conjugated with the aldehyde groups increases the ability of MUC to induce DNAPC relative to the saturated dialdehyde glutaraldehyde.

Antioxidant capacity and oxygen radical diseases in the preterm newborn.

BACKGROUND: Bronchopulmonary dysplasia, intraventricular hemorrhage, necrotizing enterocolitis, and retinopathy of prematurity may be different manifestations of oxygen radical diseases of prematurity (ORDP). OBJECTIVE: To test the hypothesis that the antioxidant capacity of cord blood serum will predict risk of ORDP. DESIGN: An inception cohort of premature neonates was followed up from birth until discharge or death to determine if outcome was related to cord blood serum antioxidant capacity, as determined by a manual assay measuring the relative inhibition of oxidation of 2,2'-azino-di-(3-ethylbenzthiazoline)-6 sulfonic acid (ABTS). Possible correlations between antioxidant capacity and various perinatal factors were also tested. SETTING: Level 3 newborn intensive care unit. PATIENTS: All inborn very low-birth-weight neonates from whom cord blood was available and for whom maternal consent was obtained were included. Newborns who died in the first week of life or who had major congenital malformations were excluded. A convenience sample of newborns weighing more than 1500 g was used to perfect assay and explore confounders. MAIN OUTCOME MEASURES: Significant ORDP was defined as the presence of intraventricular hemorrhage greater than grade 2, retinopathy of prematurity greater than stage 1, bronchopulmonary dysplasia at the postconceptional age of 36 weeks, or necrotizing enterocolitis with the hypothesis that neonates with ORDP will have lower antioxidant capacity in cord blood serum. RESULTS: Serum antioxidant capacity at birth correlated with gestational age for the entire sample of 41 neonates and for the 26 neonates born before 32 weeks' gestation. After correction for gestational age, cord serum antioxidant capacity did not correlate with maternal smoking, preeclampsia, chorioamnionitis, cord pH Apgar scores, or any of the ORDP studied. CONCLUSION: Cord serum antioxidant capacity correlates with gestational age but does not predict ORDP risk.

Effect of CYP2E1 induction by ethanol on the immunotoxicity and genotoxicity of extended low-level benzene exposure.

Potential additive effects of ethanol consumption, a common life-style factor, and low-level benzene exposure, a ubiquitous environmental pollutant, were investigated. Ethanol is a potent inducer of the cytochrome P-450 2E1 (CYP2E1) enzyme, which bioactivates benzene to metabolites with known genotoxicity and immunotoxicity. A liquid diet containing 4.1% ethanol was used to induce hepatic CYP2E1 activity by 4-fold in female CD-1 mice. Groups of ethanol-treated or pair-fed control mice were exposed to benzene or filtered air in inhalation chambers for 7 h/d, 5 d/wk for 6 or 11 wk. The initial experiment focused on immunotoxicity endpoints based on literature reports that ethanol enhances high-dose benzene effects on spleen, thymus, and bone marrow cellularity and on peripheral red blood cell (RBC) and white blood cell (WBC) counts. No statistically significant alterations were found in spleen lymphocyte cellularity, subtype profile, or function (mitogen-induced proliferation, cytokine production, or natural killer cell lytic activity) after 6 wk of ethanol diet, 0.44 ppm benzene exposure, or both. This observed absence of immunomodulation by ethanol alone, a potential confounding factor, further validates our previously established murine model of sustained CYP2E1 induction by dietary ethanol. Subsequent experiments involved a 10-fold higher benzene level for a longer time of 11 wk and focused on genotoxic endpoints in known target tissues. Bone marrow and spleen cells were evaluated for DNA-protein cross-links, a sensitive transient index of genetic damage, and spleen lymphocytes were monitored for hprt-mutant frequency, a biomarker of cumulative genetic insult. No treatment-associated changes in either genotoxic endpoint were detected in animals exposed to 4.4 ppm benzene for 6 or 11 wk with or without coexposure to ethanol. Thus, our observations suggest an absence of genetic toxicity in CD-1 mice exposed to environmentally relevant levels of benzene with or without CYP2E1 induction.

DNA-protein cross-link levels in bone marrow cells of mice treated with benzene or trans,trans-muconaldehyde.

Increased levels of DNA-protein cross-links (DNAPC) have been observed in vitro and in vivo following treatment with a number of chemotherapeutic alkylating agents and topoisomerase II inhibitors, that is, agents that have also been associated with the development of bone marrow depression and acute myelogenous leukemia. The current studies were undertaken to examine the effect of benzene, a bone marrow toxin and human leukemogen, on DNAPC levels in mouse bone marrow cells. Using a K+/sodium dodecyl sulfate (SDS) precipitation assay for DNAPC determination, the results indicate increased DNA-protein cross-link levels in mouse bone marrow cells at 2 and 4 but not 8 h after a single ip injection of 440 mg/kg benzene. Following the administration of multiple hematotoxic benzene doses (440 or 880 mg/kg, 2x/d for 2 d), increases in DNA-protein cross-link levels were either slight or not present. These results suggest that DNAPC induced by benzene are neither cumulative nor persistent lesions. The toxicity of benzene is mediated by a number of number of ring-hydroxylated and ring-opened compounds; therefore the present studies also examined DNAPC levels in mice administered trans,trans-muconaldehyde (MUC), a ring-opened hematotoxic and genotoxic metabolite of benzene. No marked increases in DNAPC levels were observed in CD- mouse bone marrow cells 1-12 h following a single ip injection of 3 mg/kg muconaldehyde. It is possible that multiple doses of MUC are required to induce elevated DNAPC levels in bone marrow cells of mice, since multiple doses are required for MUC-induced hematotoxicity. Other reactive metabolites and/or an interaction of reactive intermediates may also be involved in DNAPC induced by benzene.

Potential biomarkers of benzene exposure.

Biological markers or biomarkers of exposure are indicators for the evaluation of the internal dose of a xenobiotic. Biomarkers integrate exposure from all routes and sources. This review presents a short overview of potential biomarkers of benzene exposure currently under investigation, the methodology used for their determination, and experimental findings and their usefulness and specificity in assessing exposure to benzene. Potential biomarkers of benzene exposure are benzene, benzene metabolites, and adducts formed by reactive benzene metabolites with cellular constituents. The potential biomarkers of benzene exposure described in this review are: (1) benzene, the parent hydrocarbon; (2) ring-hydroxylated urinary metabolites, phenol, catechol, hydroquinone, and 1,2,4-trihydroxybenzene; (3) trans,trans-muconic acid, a urinary ring-opened metabolite; (4) N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine, a urinary metabolite of benzene, phenol, and hydroquinone; (5) S-phenylmercapturic acid, a glutathione-derived adduct; (6) N7-phenylguanine, a DNA adduct; and (7) S-phenylcysteine and N-phenyl-valine, hemoglobin/protein-derived adducts.

Increased gene expression in human promyeloid leukemia cells exposed to trans,trans-muconaldehyde, a hematotoxic benzene metabolite.

The hematotoxicity of benzene, a human leukemogen, has been postulated to be mediated by reactive metabolites and involve cell damage caused by reactive oxygen species. Because expression of the transcription factors AP-1 and NF-kappaB is sensitive to the redox state in eukaryotic cells, the DNA binding activity of AP-1 and NF-kappaB was examined in HL-60 promyeloid leukemia cells exposed to trans,trans-muconaldehyde, a microsomal hematotoxic metabolite of benzene. There was little AP-1 binding activity in nuclear extracts from control HL-60 cells based on electrophoretic mobility shift assays. Exposure to 0.1 microM MUC for 4 h resulted in significantly increased levels of nuclear protein with high sequence specificity for the consensus AP-1 sequence. In addition, electrophoretic mobility shift assays showed a strong increase in the binding of a factor to the NF-kappaB site. The latter was highest in nuclear extracts from HL-60 cells treated with 1.0 microM muconaldehyde and cultured for 4 h. Exposure of HL-60 cells to muconaldehyde resulted in an increase in c-fos and c-jun mRNA levels. Western blot analysis showed that the protein levels of c-jun increased in HL-60 cells treated with 1 microM muconaldehyde and cultured for 4-6 h and subsequently decreased gradually. Increased AP-1 binding was observed in bone marrow cells from B6C3F1 mice 2 h after administration of 440 mg/kg benzene. We suggest that increased gene expression of NF-kappaB and AP-1 binding activity and up-regulation of c-fos and c-jun may play a role in the mechanism of benzene leukemogenesis.

Structure-activity relationships in the hydrolysis of acrylate and methacrylate esters by carboxylesterase in vitro.

Acrylate esters are important chemicals in the plastics industry, whose toxicity is theorized to involve alkylation of critical cellular nucleophiles via the Michael addition. Carboxylesterase-mediated hydrolysis of acrylates may be a detoxification mechanism as the unsaturated acid produced is not electrophilic under physiological conditions. Using purified porcine liver carboxylesterase, the enzymatic hydrolysis of several acrylate esters was characterized to determine Km and Vmax values for each ester. The Km (microM) and Vmax (nmol/min) values observed for ethyl acrylate were 134 +/- 16 (S.D.) and 8.9 +/- 2.0, respectively. While the Km for ethyl methacrylate was not significantly different, the Vmax 5.5 +/- 2.5, was significantly lower compared with the corresponding value for ethyl acrylate. The Km and Vmax for butyl acrylate were 33.3 +/- 8.5 microM and 1.49 +/- 0.83 nmol/min, respectively, and the corresponding values for its alpha-methyl analog were not significantly different. The Km and Vmax for tetraethyleneglycol dimethacrylate were 39 +/- 15 microM and 2.9 +/- 1.0 nmol/min, respectively. The Vmax for ethyleneglycol dimethacrylate, 6.9 +/- 2.4 nmol/min, was significantly higher than that of the larger bifunctional ester tetraethyleneglycol dimethacrylate, but the Km was not significantly different. These results indicate that alpha-methyl substitution appears to have a minor effect in the enzymatic hydrolysis of acrylates, and suggest that the relative toxicity of acrylates is not due to differences in carboxylesterase-mediated hydrolysis.

Distribution studies in CD-1 mice administered [14C]muconaldehyde.

Trans,trans-muconaldehyde (muconaldehyde, MUC), a microsomal hematotoxic ring-opened metabolite of benzene, has been proposed to play a role in benzene hematotoxicity. In the present study, [14C]muconaldehyde was administered to CD-1 mice and the distribution of [14C]muconaldehyde equivalents was investigated. The study was carried out to evaluate whether [14C]muconaldehyde equivalents could reach the bone marrow. [14C]Muconaldehyde at a dose of 2 mg/kg was administered intraperitoneally (3.4 microCi/mouse) and by intravenous injection (2.7 microCi/mouse). The amount of [14C]muconaldehyde equivalents was measured in the bone marrow, blood, liver, lung, kidney and spleen at 0.25, 0.5, 1, 2, 4, and 24 h after [14C]MUC administration. The results indicate that 0.044% or 0.018% of the total dose administered when given i.v. or i.p., respectively, reached the bone marrow. The elimination of the radioactivity in all organs had at least two phases. The bone marrow, kidney, and lung had a rapid first phase (t1/2 0.5-1.2 h) and a slower second phase (t1/2 2.8-15.7 h). In the liver, a slow first phase (t1/2 3.7 h) was followed by a more rapid second phase (t1/2 1.5 h). The level of radioactivity in blood and bone marrow was significantly higher when [14C]muconaldehyde was administered intravenously compared with intraperitoneally, demonstrating that the route of administration affects the distribution of [14C]muconaldehyde equivalents.

Reactive ring-opened aldehyde metabolites in benzene hematotoxicity.

The hematotoxicity of benzene is mediated by reactive benzene metabolites and possibly by other intermediates including reactive oxygen species. We previously hypothesized that ring-opened metabolites may significantly contribute to benzene hematotoxicity. Consistent with this hypothesis, our studies initially demonstrated that benzene is metabolized in vitro to trans-trans-muconaldehyde (MUC), a reactive six-carbon diene dialdehyde, and that MUC is toxic to the bone marrow in a manner similar to benzene. Benzene toxicity most likely involves interactions among several metabolites that operate by different mechanisms to produce more than one biological effect. Our studies indicate that MUC coadministered with hydroquinone is a particularly potent metabolite combination that causes bone marrow damage, suggesting that the involvement of ring-opened metabolites in benzene toxicity may be related to their biological effects in combination with other benzene metabolites. Studies in our laboratory and by others indicate that MUC is metabolized to a variety of compounds by oxidation or reduction of the aldehyde groups. The aldehydic MUC metabolite 6-hydroxy-trans-trans-2,4-hexadienal (CHO-M-OH), similar to MUC but to a lesser extent, is reactive toward glutathione, mutagenic in V79 cells, and hematotoxic in mice. It is formed by monoreduction of MUC, a process that is reversible and could be of biological significance in benzene bone marrow toxicity. The MUC metabolite 6-hydroxy-trans-trans-2,4-hexadienoic (COOH-M-OH) is an end product of MUC metabolism in vitro. Our studies indicate that COOH-M-OH is a urinary metabolite of benzene in mice, a finding that provides further indirect evidence for the in vivo formation of MUC from benzene. Mechanistic studies showed the formation of cis-trans-muconaldehyde in addition to MUC from benzene incubated in a hydroxyl radical-generating Fenton system. These results suggest that the benzene ring is initially opened to cis,cis-muconaldehyde, an unstable isomer that rearranges to cis-trans-muconaldehyde, which further rearranges to trans-trans-muconaldehyde. The latter is not formed from benzene dihydrodiol by reactive oxygen species in a Fenton system that contains reactive oxygen species.

Iron-stimulated ring-opening of benzene in a mouse liver microsomal system. Mechanistic studies and formation of a new metabolite.

In the present study, we investigated the mechanism(s) of ring-opening of benzene in a mouse liver microsomal system in the presence of Fe2+.HPLC analysis based on coelution with authentic standards and on-line UV spectra obtained using a diode array detector indicated that benzene is metabolized to phenol, hydroquinone (HQ), trans,trans-muconaldehyde (muconaldehyde, MUC), 6-oxo-trans,trans-2,4-hexadienoic (COOH-M-CHO), 6-hydroxy-trans,trans-2,4-hexadienal (CHO-M-OH), and 6-hydroxy-trans,trans-2,4-hexadienoic acid (COOH-M-OH). CHO-M-OH was confirmed by mass spectrometry. Muconaldehyde was also metabolized to CHO-M-OH, COOH-M-CHO and COOH-M-OH, in the same microsomal system. The inhibition of muconaldehyde metabolism by microsomes in the presence of pyrazole indicates that there is cytosolic alcohol dehydrogenase (ADH) activity in the microsomes. Metabolism by contaminating ADH of muconaldehyde formed during microsomal incubation of benzene could be involved in the formation of CHO-M-OH and COOH-M-OH. The ring-opening of benzene was stimulated by added Fe2+. Hydrogen peroxide was produced in the microsomal system and consumed in the presence of added Fe2+. Addition of catalase inhibited the formation of ring-opened products, while superoxide dismutase increased their formation in the presence of azide. Singlet oxygen scavengers, i.e. histidine, deoxyguanosine, Tris and azide (at concentrations above 1.0 mM), dramatically decreased the ring-opening of benzene. Hydroxyl radical scavengers, DMSO, mannitol and formate, but not ethanol, also decreased the ring-opening of benzene. The data indicate that Fenton chemistry plays an important role in benzene ring-opening by microsomes. An unknown peak with UV absorption maxima at 275 and 345 nm was also detected. Based on pH sensitivity of the UV spectrum, the reactivity with thiobarbituric acid (giving a chromogen with absorption maximum at 532 nm) and the molecular weight (126), this compound was identified tentatively as alpha- or beta-hydroxymuconaldehyde.

The hematotoxic effects of 6-hydroxy-trans,trans-2,4-hexadienal, a reactive metabolite of trans,trans-muconaldehyde, in CD-1 mice.

6-Hydroxy-trans,trans-2,4-hexadienal (CHO-M-OH) is a metabolite of trans,trans-muconaldehyde (muconaldehyde or MUC), a microsomal hematotoxic ring-opened metabolite of benzene. In the present study, the toxicity of CHO-M-OH was examined. In order to assess potential toxic effects of CHO-M-OH on the maturation of erythroid cells in the bone marrow, 10-week-old male CD-1 mice were administered CHO-M-OH intraperitoneally and 59Fe incorporation into erythrocytes was measured. The uptake of 59Fe by erythroid cells was significantly inhibited at doses of 20, 25, and 30 mg/kg. There was no inhibition of 59Fe incorporation at a dose of 15 mg/kg. In other hematotoxicity studies, bone marrow cellularity, peripheral blood cells, and sulfhydryl contents in bone marrow cells were examined in mice administered CHO-M-OH intraperitoneally. An increase in the white blood cell count was observed in mice treated with 5 mg/kg/day for 16 consecutive days, while bone marrow cellularity and red blood cell parameters were not changed. Administration of 10 mg/kg/day for 16 consecutive days caused a significant decrease in sulfhydryls of bone marrow cells but no changes in bone marrow cellularity and peripheral blood parameters compared with controls. At a dose of 25 mg/kg/day for 4 days, there was a significant decrease in nucleated bone marrow cells. The white blood cell count, mainly lymphocytes, also significantly decreased. Our results indicate that CHO-M-OH is a hematotoxin in mice and conceivably could play a role in benzene toxicity.

Studies on pathways of ring opening of benzene in a Fenton system.

Ring-opened products of benzene metabolism have been postulated to play a role in hematotoxicity and leukemogenesis. The reaction of benzene in the Fenton system was reexamined to determine the presence of compounds which might serve as intermediates in the formation of trans, trans-muconaldehyde (MUC), a microsomal hematotoxic metabolite of benzene. Benzene dihydrodiol (DHD) was found in this system based on coelution with authentic standard, ultraviolet (UV) absorption characteristics, and molecular weight. Incubation of DHD in the Fenton system resulted in the formation of phenol (PH), catechol (CAT), and products which reacted with thiobarbituric acid to form chromogens absorbing at 495 nm and 532 nm, consistent with products containing an alpha, beta-unsaturated aldehyde group. However, muconaldehyde was not detected in the Fenton system incubated with DHD, indicating that MUC is not formed via ring opening of DHD. When benzene was incubated in the Fenton system, MUC, cis,trans-muconaldehyde, PH, hydroquinone (HQ), and CAT were identified. Identification of cis,trans-muconaldehyde, an isomer which can quickly rearrange to MUC, suggests that cis,cis-muconaldehyde is originally formed from benzene and converted to cis,trans- and then trans,trans-muconaldehyde.

Evaluation of assays for the identification and quantitation of muconic acid, a benzene metabolite in human urine.

Muconic acid (MA) is a urinary metabolite of benzene and has been used as a biomarker of exposure to benzene in humans exposed to levels as low as 1 ppm. We have modified a high-pressure liquid chromatography (HPLC) based assay for urinary MA (Ducos et al., 1990) by the use of a diode array detector. This modification increases the specificity of the HPLC-based assay by identifying false positives. In addition, we have developed a gas chromatography (GC) based assay that uses a flame ionization detector (GC-FID). Both assays identified and quantified MA in human urine at concentrations greater than 40-50 ng/ml. Assay precision was within 10% relative standard deviation for MA concentrations above 90 ng/ml using the HPLC assay and above 40 ng/ml using the GC-FID assay. Quantitative accuracy of the assays was evaluated by determining MA in human urine samples using both methods and also a gas chromatography-mass spectrometry (GC-MS) procedure. Numerical correlation among the three assays was good at MA concentrations above 100 ng/ml.

The reactivity of selected acrylate esters toward glutathione and deoxyribonucleosides in vitro: structure-activity relationships.

Acrylate esters are alpha,beta-unsaturated esters used as plastic monomers whose toxicity may involve reaction with tissue nucleophiles via Michael addition. Structure-activity relationships for reactivity of selected esters with glutathione (GSH) and deoxyribonucleosides were investigated in the present studies. The esters investigated were methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, butyl acrylate, butyl methacrylate, tetraethyleneglycol diacrylate, tetraethyleneglycol dimethacrylate, and ethyleneglycol dimethacrylate. To compare their reactivities toward GSH, esters were incubated for up to 1 hr at 37 degrees C and pH 7.4 with either GSH or red blood cells in phosphate-buffered saline followed by measurement of free thiol. In both systems acrylate electrophilic reactivity decreased with alpha-methyl substitution; however, the decrease in electrophilic reactivity was more evident in the cell-free system than in the red blood cell model. Increased alcohol chain length moderately affected the apparent second-order rate constant for the spontaneous reaction of acrylate esters with GSH, but did not affect potency relative to cellular GSH depletion. The apparent second-order rate constants of bifunctional esters are more than twice the rate constants of the much smaller monofunctional esters. Ethyl acrylate, a reactive acrylate ester based upon glutathione alkylation, has been designated a class 2B (suspect human) carcinogen by the International Agency for Research on Cancer. To detect possible DNA alkylation by acrylate esters in vitro, ethyl acrylate was incubated with deoxyribonucleosides for up to 24 hr at pH 6.7 or 7.4 and 37 degrees C or up to 8 hr and 50 degrees C. HPLC analysis revealed no detectable adduct formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of trans,trans-muconaldehyde with bovine serum albumin.

Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that incubation of bovine serum albumin (BSA) with 10 microM-1.0 mM trans,trans-muconaldehyde results in the formation of a new band with molecular weight 105 kDa as well as high-molecular-weight material (> 200 kDa), suggesting intermolecular cross-linking of BSA by muconaldehyde. Muconaldehyde-reacted BSA exhibits a decrease in amino groups as measured by the fluorescamine assay. Spectroscopic analysis of the BSA-muconaldehyde incubation mixture shows the formation of two new peaks with absorption maxima at 340 and 475 nm. Gel filtration chromatography on Sephadex G-200 of muconaldehyde-reacted BSA shows elution of a high-molecular-weight fraction and a second fraction which elutes at the elution volume of monomeric unreacted BSA. Both fractions contain material which absorbs light at 280 nm (protein absorption), as well as at 340 and 475 nm, while chromatographed fractions containing unreacted BSA show absorption at 280 nm only. When excited at 340 nm, fractions of muconaldehyde-reacted BSA also exhibit fluorescence emission with a maximum at about 430 nm, whereas excitation at 475 nm does not result in fluorescence emission. Incubation of BSA with the aldehydic muconaldehyde metabolites trans,trans-6-oxo-hexadienoic acid and trans,trans-6-hydroxy-hexa-2,4-dienal or the corresponding diacid trans,trans-muconic acid did not cause any of the effects described above for muconaldehyde, suggesting that the diene-dialdehyde structure of muconaldehyde is a requirement for cross-linking and for the formation of the fluorescing chromophore.