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.

Biological interactions of alpha,beta-unsaturated aldehydes.

This article describes the chemical nature of alpha,beta-unsaturated aldehydes and some of their toxicological effects based on their ability to function as direct-acting alkylating agents. Selected compounds discussed include alpha,beta-unsaturated aldehydic environmental pollutants, metabolites of xenobiotics and natural products, and lipid peroxidation--and DNA oxidation products derived from cellular constituents. Briefly reviewed are sources and mechanisms of formation of the aldehydes, their reactivity with respect to glutathione and amino-groups, their toxicity based on interaction with sulfhydryl and amino targets in cells, and modulation of their toxicity by metabolism.

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.

Tumor promoters stimulate the formation of 1,2-diacylglycerol in murine peritoneal macrophages: a possible mechanism for stimulating superoxide anion radical production.

The formation of 1,2-diacylglycerol (DAG), a known stimulator of superoxide anion radical (O2-.) production in inflammatory cells, was assessed in murine peritoneal macrophages following treatment in vitro with tumor promoters. Addition of phorbol-12-myristate-13-acetate (PMA, 1-100 ng/ml) to resident peritoneal macrophage cultures from CD-1 female mice resulted in a 3- to 7-fold increase in [3H]DAG formation. The response was observed from 15 min to 2 h following the addition of PMA. At concentrations at which they stimulate O2-. production, PMA and other tumor promoters such as mezerein, phorbol-12,13-dibutyrate and 4-O-methyl-PMA stimulated the formation of [3H]DAG. Similar results were obtained when thioglycollate-elicited macrophages were used. Concurrent with the formation of [3H]DAG was a release of [3H]choline equivalents from the resident peritoneal macrophages treated with tumor promoters. The calcium ionophore A23187 did not stimulate O2-. production of [3H]DAG formation in resident peritoneal macrophages. These results demonstrate that tumor promoters stimulate the accumulation of DAGs in murine peritoneal macrophages at concentrations at which they stimulate O2-. production and suggest a mechanism by which tumor promoters such as mezerein, which are weak activators of protein kinase C, may indirectly stimulate O2-. production.

Stimulation of human polymorphonuclear leukocyte superoxide anion radical production by tumor promoters.

Comparison was made of the ability of the potent tumor promoter phorbol myristate acetate (PMA), as well as less active PMA analogs and non-phorbol ester tumor promoters, to stimulate superoxide anion radical (O-.2) production by human polymorphonuclear leukocytes (PMN). The rate of O-.2 production was found to correlate with the tumor-promoting activity of the phorbol esters as opposed to their inflammatory activity. Mezerein and telocidin B were slightly better stimulators of O-.2 production than PMA. Acetic acid was inactive. These data are discussed in terms of a possible role for O-.2 and other reactive oxygen species in tumor promotion.

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.

Inhibition of microsomal cytochrome c reductase activity by a series of alpha, beta-unsaturated aldehydes.

alpha, beta-Unsaturated aldehydes are reactive and cytotoxic compounds which occur in the environment and are also formed in vivo. Many of these aldehydes have been reported to inhibit hepatic cytochrome P-450. Our laboratory has shown that trans,trans-muconaldehyde (a possible metabolite of benzene) as well as acrolein and crotonaldehyde, when added to hepatic microsomes, decreased cytochrome P-450 (measured spectrophotometrically). Additional studies showed that several alpha, beta-unsaturated aldehydes also inhibited hepatic microsomal NADPH-cytochrome c reductase. Acrolein, crotonaldehyde and trans,trans-muconaldehyde all decreased NADPH-cytochrome c reductase activity in vitro. Concentrations of 0.5, 1.0 and 1.5 mM acrolein decreased activity to 60, 26 and 11% of control respectively. Similar concentrations of trans,trans-muconaldehyde inhibited NADPH-cytochrome c reductase. Crotonaldehyde was a less effective inhibitor of this enzyme. Propionaldehyde, a saturated aldehyde, had no effect on NADPH-cytochrome c reductase activity. Time course experiments with acrolein over a period of 5-45 min suggest that the loss of NADPH-cytochrome c reductase activity is non-linear. The addition of reduced glutathione protected against the inhibition of reductase activity by acrolein. Formation of these aldehydes and their subsequent inhibition of these enzymes may have important consequences in xenobiotic metabolism.

Comparative metabolism of benzene and trans,trans-muconaldehyde to trans,trans-muconic acid in DBA/2N and C57BL/6 mice.

Our laboratory recently identified trans,trans-muconaldehyde (MUC), a six-carbon diene dialdehyde, as a hematotoxic microsomal metabolite of benzene (Latriano et al., Proc Natl Acad Sci USA 83: 8356-8360, 1986). We also showed that MUC is metabolized in vitro to trans,trans-muconic acid (MA), a six-carbon diene dicarboxylic acid and known urinary metabolite of benzene. To elucidate further the role of ring-opened metabolites in benzene toxicity, the metabolism of benzene and MUC was examined in the benzene sensitive DBA/2N mouse strain and the less benzene sensitive C57BL/6 strain. A sensitive assay for urinary MA analysis was developed. The percent of benzene dose excreted as urinary MA within the first 24 hr after treatment decreased with an increase in benzene dose, i.e. from 9.8 to 0.4% in DBA/2N mice and from 17.6 to 0.2% in C57BL/6 mice treated with 0.5 to 880 mg/kg benzene. DBA/2N mice excreted significantly (P less than or equal to 0.05) more MA compared with C57BL/6 mice after treatment with hematotoxic benzene doses (220-880 mg/kg). At low benzene doses (0.5 to 2.5 mg/kg), C57BL/6 mice excreted significantly (P less than or equal to 0.05) more MA compared with DBA/2N mice. There were no significant differences in the metabolism of MUC to MA between the two strains after treatment with 0.5 to 3.0 mg/kg. Furthermore, mice from both strains excreted similar amounts of muconic acid when treated with 0.7 to 7.1 mg/kg MA. These results are consistent with the hypothesis that reactive ring-opened metabolites such as trans,trans-muconaldehyde play a role in benzene hematotoxicity. Sensitivity towards benzene may be due, in part, to increased metabolism to ring-opened compounds.

Inhibition by reactive aldehydes of superoxide anion radical production from stimulated polymorphonuclear leukocytes and pulmonary alveolar macrophages. Effects on cellular sulfhydryl groups and NADPH oxidase activity.

Alpha,beta-unsaturated aldehydes such as acrolein (ACR) and crotonaldehyde (CRO) have been shown previously in our laboratory to inhibit the production of superoxide anion radical (O2-) by stimulated phagocytic cells in vitro in a dose-related manner. Based on the known reactivity of these compounds towards cellular sulfhydryls (SH), the present studies were aimed at investigating cellular SH status in relation to O2- production. Plasma membrane surface SH groups were measured using carboxypyridinedisulfide and monitoring the resultant formation of mixed disulfides through assay of thione released into the supernatant fraction. Intracellular non-protein sulfhydryls were measured using 5,5'-dithiobis-2-nitrobenzoic acid. In both human polymorphonuclear leukocytes (PMN) and rat pulmonary alveolar macrophages (PAM) there was a dose-related decrease in surface SH and soluble SH after ACR and CRO treatment. Propionaldehyde, a three-carbon saturated aldehyde, was without effect. The decrease in surface SH was greater than the decrease in soluble SH. In addition, in PMN and PAM preincubated with 5-40 microM ACR, there was a dose-related inhibition in the rate of O2- production with no effect on the lag time as measured by cytochrome c reduction. In stimulated PMN, there was a dose-related decrease in the rate after addition of 5-40 microM ACR. These data suggest that changes in SH status by reactive aldehydes can modulate the activity of the plasma membrane NADPH oxidase responsible for O2- production.

The role of free radicals in tumor promotion.

A role has been suggested for free radicals and active states of oxygen in tumor promotion. There are a number of lines of support for this hypothesis, but no definitive evidence. The hypothesis has proven of value in leading to the development of models pertinent to understanding the mechanism of action of tumor promoters.

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.

Metabolism and toxicity of trans,trans-muconaldehyde, an open-ring microsomal metabolite of benzene.

We have previously hypothesized that ring-opened metabolites may play an important role in benzene toxicity. In this paper we review recent work related to this hypothesis. trans,trans-Muconaldehyde (TTM), a six-carbon diene dialdehyde, was shown by our laboratory to be a microsomal metabolite of benzene. This compound is a ring-opened metabolite of benzene that is hematotoxic in mice. The toxicity of TTM may stem in part from its ability to act as a direct-acting alkylating agent involving interaction with cellular sulfhydryls and/or amino groups. On the other hand, metabolism to the diacid trans,trans-muconic acid (MA), a known urinary metabolite of benzene, may represent detoxification since this results in loss of electrophilicity of the compound. Preliminary results indicate that TTM can be metabolized to MA in vitro and in vivo. The interaction of TTM in vitro with macrophages and neutrophils, key cells in the bone marrow, results in cell membrane changes, including loss of activity in the plasma membrane-bound NADPH-dependent oxidase and decreases in membrane lipid fluidity. Deoxyguanosine also was found to react with TTM, forming several different products. These findings may be due to TTM acting directly as an alkylating agent.

The toxicology of benzene.

Benzene is metabolized, primarily in the liver, to a series of phenolic and ring-opened products and their conjugates. The mechanism of benzene-induced aplastic anemia appears to involve the concerted action of several metabolites acting together on early stem and progenitor cells, as well as on early blast cells, such as pronormoblasts and normoblasts to inhibit maturation and amplification. Benzene metabolites also inhibit the function of microenvironmental stromal cells necessary to support the growth of differentiating and maturing marrow cells. The mechanism of benzene-induced leukemogenesis is less well understood. Benzene and its metabolites do not function well as mutagens but are highly clastogenic, producing chromosome aberrations, sister chromatid exchange, and micronuclei. Benzene has been shown to be a multi-organ carcinogen in animals. Epidemiological studies demonstrate that benzene is a human leukemogen. There is need to better define the lower end of the dose-response curve for benzene as a human leukemogen. The application of emerging methods in biologically based risk assessment employing pharmacokinetic and mechanistic data may help to clarify the uncertainties in low-dose risk assessment.

Chromatographic and spectrophotometric characterization of adducts formed during the reaction of trans,trans-muconaldehyde with 14C-deoxyguanosine 5'-phosphate.

Mice liver microsomes oxidatively open the benzene ring to form trans,trans-muconaldehyde, a hematotoxic unsaturated aldehyde. In the present studies, 4.5 mumole trans,trans-muconaldehyde was reacted with 14C-2'deoxyguanosine 5'-phosphate in phosphate buffer. Products were separated by high performance liquid chromatography (HPLC). Absorbance was monitored using a diode array detector, and aliquots of the HPLC eluant were collected for UV spectrophotometric analysis and scintillation counting. Under these conditions, deoxyguanosine 5'-phosphate eluted at 12.5 min and muconaldehyde at 22.0 min. The HPLC and radioactivity profiles of the muconaldehyde/deoxyguanosine reaction mixture indicated the presence of multiple adducts. Three adducts were detected eluting at 36, 39, and 42 min, which represented approximately 2.5, 2.5, and 1% of the radioactivity, respectively. These adducts had similar UV spectra with absorption maxima between 334 and 347 nm. Another product of the reaction mixture, eluting at 19.0 min and accounting for 10% of the radioactivity, was also observed. This compound had absorption maxima at 348 and 372 nm. These results suggest that trans,trans-muconaldehyde can react with deoxyguanosine monophosphate in vitro under physiological conditions to form stable adducts. Studies are being conducted to determine the structure of these adducts and whether these adducts are formed by the reaction of DNA with muconaldehyde or metabolically activated benzene.

Identification of 6-hydroxy-trans,trans-2,4-hexadienoic acid, a novel ring-opened urinary metabolite of benzene.

We studied the in vivo metabolism of benzene in mice to ring-opened compounds excreted in urine. Male CD-1 mice were treated intraperitoneally with benzene (110-440 mg/kg), [14C]benzene (220 mg/kg) or trans, trans-muconaldehyde (MUC; 4 mg/kg), a microsomal, hematotoxic metabolite of benzene. Urine, collected over 24 hr, was extracted and analyzed by HPLC with a diode-array detector and by scintillation counting. In addition to trans,trans-muconic acid, previously the only known ring-opened urinary benzene metabolite, a new metabolite, 6-hydroxy-trans,trans-2,4-hexadienoic acid, was detected in urine of mice treated with either benzene or MUC. We identified the new metabolite based on coelution of metabolites and UV spectral comparison with authentic standards in unmethylated and methylated urine extracts. Results presented here are consistent with the intermediacy of MUC in the in vivo metabolism of benzene to ring-opened metabolites.

Studies on the mechanism of benzene toxicity.

Using the 59Fe uptake method of Lee et al. it was shown that erythropoiesis in female mice was inhibited following IP administration of benzene, hydroquinone, p-benzoquinone, and muconaldehyde. Toluene protected against the effects of benzene. Coadministration of phenol plus either hydroquinone or catechol resulted in greatly increased toxicity. The combination of metabolites most effective in reducing iron uptake was hydroquinone plus muconaldehyde. We have also shown that treating animals with benzene leads to the formation of adducts of bone marrow DNA as measured by the 32P-postlabeling technique.

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.

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.

Mutagenicity of trans,trans-muconaldehyde and its metabolites in V79 cells.

trans,trans-Muconaldehyde (MUC), a six-carbon-diene-dialdehyde, is a microsomal, hematotoxic ring-opened metabolite of benzene. MUC is metabolized to a variety of compounds which are formed by oxidation and/or reduction of the aldehyde group(s). In the present studies, MUC and its metabolites were examined for mutagenic activity at the hypoxanthine guanine phosphoribosyltransferase (HGPRT) locus in Chinese hamster V79 cells. Mutagenicity was scored by counting 8-azaguanine-resistant colonies. Of the 6 compounds tested, MUC and its aldehydic metabolites 6-hydroxy-trans,trans-2,4-hexadienal and 6-oxo-trans,trans-hexadienoic acid were mutagenic in that order of potency. The other MUC metabolites tested (1,6-dihydroxy-trans, trans-2, 4-hexadiene, trans, trans-muconic acid, and 6-hydroxy-trans, trans-2,4-hexadienoic acid) had little or not activity in this system. The order of mutagenic activity of MUC and its aldehydic metabolites correlates with their reactivity towards glutathione, suggesting that alkylating potential is important in the genotoxicity of these compounds.

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.