Protein adducts of 1,4-benzoquinone and benzene oxide among smokers and nonsmokers exposed to benzene in China.

Hemoglobin (Hb) and albumin (Alb) adducts of the benzene metabolites benzene oxide (BO) and 1,4-benzoquinone (1,4-BQ) were analyzed by gas chromatography-mass spectrometry in 43 exposed workers and 44 unexposed controls from Shanghai, China, as part of a larger cross-sectional study of benzene biomarkers. When subjects were divided into controls (n = 44) and workers exposed to 31 ppm (n = 22) of benzene, median 1,4-BQ-Alb adducts were 2110, 5850, and 13,800 pmol/g Alb, respectively (correlation with exposure: Spearman r = 0.762; P < 0.0001); median BO-Alb adducts were 106, 417, and 2400 pmol/g Alb, respectively (Spearman r = 0.877; P < 0.0001); and median BO-Hb adducts were 37.1, 50.5, and 136 pmol/g Hb, respectively (Spearman r = 0.757; P < 0.0001). To our knowledge, this is the first observation that adducts of 1,4-BQ are significantly correlated with benzene exposure. When compared on an individual basis, Alb adducts of 1,4-BQ and BO and Hb adducts of BO were highly correlated with each other and with urinary phenol and hydroquinone (P < 0.0001 for all of the comparisons). Although detectable in the assays, Hb adducts of 1,4-BQ and both Hb and Alb adducts of 1,2-BQ produced erratic results and are not reported. Interestingly, cigarette smoking increased Alb adducts of 1,4-BQ but not of BO, suggesting that benzene from cigarette smoke was not the primary contributor to the 1,4-BQ adducts.

Protein adducts as dosimeters of human exposure to styrene, styrene-7,8-oxide, and benzene.

Cysteinyl adducts of hemoglobin (Hb) and albumin (Alb) formed via reactions with reactive species were measured in 48 subjects exposed to styrene (0.24-55.2 ppm) and to styrene-7,8-oxide (SO) (2.65-107 ppb) in a factory producing boats in the USA. Hb and Alb adducts were also investigated among 88 workers exposed to benzene (0-138 ppm) in several Chinese factories. The particular adducts were S-(2-hydroxy-1-phenylethyl) cysteine, from reactions of SO with Alb (designated SO-Alb), and S-phenylcysteine, from reactions of the CYP450 benzene metabolite, benzene oxide (BO), with Hb and Alb (designated BO-Hb and BO-Alb, respectively). The relationships between adduct levels and exposures were investigated in both studies. The estimated slopes varied considerably among the particular combinations of adduct and agent to which the workers were exposed, ranging from 0.815 pmol BO-Hb/g Hb per ppm benzene to 24400 pmol SO-Alb/g Alb per ppm SO. We used these estimated slopes, along with kinetic constants, to predict the systemic doses of SO and BO in humans per mg of styrene, SO or benzene per kg body weight, under certain assumptions. Using RX to signify the particular electrophile (SO or BO) the doses of RX to the blood per unit of dose varied between 2.21 and 4110 nM RX-h/mg agent per kg b.w. The dose of RX to the blood arising from inhalation of SO was almost 2000 times that of styrene (i.e. 4110 vs. 2.21 nM RX/mg agent per kg b.w.) and 430-781 times that of benzene (i.e. 4110 vs. 5.26-9.55 nM RX/mg agent per kg b.w.), depending upon the study. Comparable estimates of the blood dose of BO were obtained from adducts of Hb and Alb and two independent studies of BO-Alb yielded similar dose estimates. These results point to the utility of protein adducts as dosimeters of reactive electrophilic species in occupational studies. Finally, significant levels of background adducts of SO and BO with Hb and Alb were observed among workers, among control subjects and in commercial human proteins. Levels of these background adducts were too great to have arisen from non-occupational exposures to styrene or benzene or from cigarette smoking.

Investigation of benzene oxide in bone marrow and other tissues of F344 rats following metabolism of benzene in vitro and in vivo.

This study examines the initial activation of benzene, exploring key aspects of its metabolism by measurement of benzene oxide (BO) and BO-protein adducts in vitro and in vivo. To assess the potential influence of various factors on the production of BO, microsomes were prepared from tissues that were either targets of benzene toxicity, i.e. the bone marrow and Zymbal glands, or not targets, i.e. liver and kidneys, of control and acetone-treated F344 rats. No BO or phenol was detected in microsomal preparations of bone marrow or Zymbal glands (less than 0.007 nmol BO/mg protein and 0.7 nmol phenol/mg protein). On the other hand, BO and phenol were readily detected in preparations of liver and kidney microsomes and acetone pretreatment resulted in a 2-fold (kidney) increase or 3.7-fold (liver) increase in production of these metabolites. Initial rates of BO production in the liver isolates were 30 (control) to 50 (acetone-treated) times higher than in the corresponding kidney tissues. The estimated half-life of BO in bone marrow homogenates was 6.0 min and the second-order reaction rate constant was estimated to be 1.35 x 10(-3) l (g bone marrow)(-1) (h)(-1). These kinetic constants were used with measurements of BO-bone marrow adducts in F344 rats, receiving a single gavage dosage of 50-400 mg benzene (kg body weight)(-1) (McDonald, T.M., et al. (1994), Cancer Res. 54, 4907-4914), to predict the bone marrow dose of BO. Among the rats receiving 400 mg (kg body weight) (-1), a BO dose of 1.13 x 10(3) nM BO-h was estimated for the bone marrow, or roughly 40% of the corresponding blood dose predicted from BO-albumin adducts. Together these data suggest that, although BO is not produced at detectable levels in the bone marrow or Zymbal glands of F344 rats, BO is rapidly distributed via the bloodstream to these tissues where it may play a role in toxicity.

A new assay for albumin and hemoglobin adducts of 1,2- and 1,4-benzoquinones.

A new method has been developed to detect mono-S-substituted cysteinyl adducts of 1,2- and 1,4-benzoquinone (BQ) in hemoglobin (Hb) and albumin (Alb). After reacting the protein with trifluoroacetic anhydride and methanesulfonic acid, the resulting isomers of O,O',S-tris-trifluoroacetyl-hydroquinone and -catechol are extracted and detected by gas-chromatography-mass spectrometry in the negative-ion chemical ionization mode. The limit of detection of the assay is about 20 pmol adduct/g protein. This assay was employed to quantitate mono-S-substituted background adducts in human and rat Hb and Alb and benzene-specific adducts in Hb and Alb from F344 rats following a single oral dosage of 50-400 mg [13C6]benzene/kg body wt. In Alb, a dose-related increase in both [13C6]1,2- and [13C6]1,4-BQ adducts was observed with [[13C6]]1,4-BQ-Alb] >> [[13C6]1,2-BQ-Alb]. The formation of [13C6]1,2-BQ-Alb was linear with increasing dosage of benzene with a slope of 2.3 (pmol adduct/g protein)/(mg/kg body wt.) (S.E. = 0.18, R2 = 0.91). However, at dosages above about 100 mg [13C6]benzene/kg body wt., the levels of 1,4-BQ-Alb were greater than proportional to the dosage. Mono-S-substituted adducts of [13C6]1,2-BQ and [13C6]1,4-BQ were not detected in Hb. The background ([12C6]) adducts of 1,2- and 1,4-BQ in 20 F344 rats were estimated (in nmol adduct/g of protein) to be 3.9 (S.E. = 0.23) and 4.9 (S.E. = 0.30) in Hb and 2.7 (S.E. = 0.24) and 11.4 (S.E. = 0.60) in Alb. At the highest dosage of 400 mg [13C6]benzene/kg body wt., background levels of 1,2-BQ-Alb were about 4-fold higher than those of the benzene-specific adducts whereas the benzene-specific levels of 1,4-BQ-Alb were about 7-fold higher than those of the background adducts. Background levels of 1,2- and 1,4-BQ adducts in 10 portions of commercial human proteins were found to be (in nmol adduct/g of protein) 1.6 (S.E. = 0.05) and 0.85 (S.E. = 0.04) in Hb and 1.6 (S.E. = 0.06) and 8.9 (S.E. = 0.36) in Alb.

Hemoglobin and albumin adducts of benzene oxide among workers exposed to high levels of benzene.

Benzene oxide (BO) reacts with cysteinyl residues in hemoglobin (Hb) and albumin (Alb) to form protein adducts (BO-Hb and BO-Alb), which are presumed to be specific biomarkers of exposure to benzene. We analyzed BO-Hb in 43 exposed workers and 42 unexposed controls, and BO-Alb in a subsample consisting of 19 workers and 19 controls from Shanghai, China, as part of a larger cross-sectional study of benzene biomarkers. The adducts were analyzed by gas chromatography-mass spectrometry following reaction of the protein with trifluoroacetic anhydride and methanesulfonic acid. When subjects were divided into controls (n = 42) and workers exposed to < or =31 (n = 21) and >31 p.p.m. (n = 22) benzene, median BO-Hb levels were 32.0, 46.7 and 129 pmol/g globin, respectively (correlation with exposure: Spearman r = 0.67, P < 0.0001). To our knowledge, these results represent the first observation in humans that BO-Hb levels are significantly correlated with benzene exposure. Median BO-Alb levels in these 3 groups were 103 (n = 19), 351 (n = 7) and 2010 (n = 12) pmol/g Alb, respectively, also reflecting a significant correlation with exposure (Spearman r = 0.90, P < 0.0001). The blood dose of BO predicted from both Hb and Alb adducts was very similar. These results clearly affirm the use of both Hb and Alb adducts of BO as biomarkers of exposure to high levels of benzene. As part of our investigation of the background levels of BO-Hb and BO-Alb found in unexposed persons, we analyzed recombinant human Hb and Alb for BO adducts. Significant levels of both BO-Hb (19.7 pmol/g) and BO-Alb (41.9 pmol/g) were detected, suggesting that portions of the observed background adducts reflect an artifact of the assay, while other portions are indicative of either unknown exposures or endogenous production of adducts.

Formation of hemoglobin and albumin adducts of benzene oxide in mouse, rat, and human blood.

Little is known about the formation and disposition of benzene oxide (BO), the initial metabolite arising from oxidation of benzene by cytochrome P450. In this study, reactions of BO with hemoglobin (Hb) and albumin (Alb) were investigated in blood from B6C3F1 mice, F344 rats, and humans in vitro. The estimated half-lives of BO in blood were 6.6 min (mice), 7.9 min (rats), and 7.2 min (humans). The following second-order rate constants were estimated for reactions between BO and cysteinyl residues of Hb and Alb [in units of L (g of Hb- or Alb-h)-1]: mouse Hb = 1.16 x 10(-)4, rat Hb = 15.4 x 10(-)4, human Hb = 0.177 x 10(-)4, mouse Alb = 2.68 x 10(-)4, rat Alb = 4.96 x 10(-)4, and human Alb = 5.19 x 10(-)4. These rate constants were used with BO-adduct measurements to assess the systemic doses of BO arising from benzene in vivo in published animal and human studies. Among rats receiving a single gavage dose of 400 mg of benzene/kg of body weight, the BO dose of 2.62 x 10(3) nM BO-h, predicted from Alb adducts, was quite similar to the reported AUC0-infinity = 1.09 x 10(3) nM BO-h of BO in blood. Interestingly, assays of Hb adducts in the same rats predicted a much higher dose of 14.7 x 10(3) nM BO-h, suggesting possible in situ generation of adducts within the erythrocyte. Doses of BO predicted from Alb adducts were similar in workers exposed to benzene [13.3 nM BO-h (mg of benzene/kg of body weight)-1] and in rats following a single gavage dose of benzene [8. 42 nM BO-h (mg of benzene/kg of body weight)-1]. Additional experiments indicated that crude isolates of Hb and Alb had significantly higher levels of BO adducts than dialyzed proteins, suggesting that conjugates of low-molecular-weight species were abundant in these isolates.

Albumin and hemoglobin adducts as biomarkers of exposure to styrene in fiberglass-reinforced-plastics workers.

OBJECTIVE: The purpose of this work was to compare levels of styrene-7,8-oxide (SO) adducts of albumin (Alb) and hemoglobin (Hb) with those of two urinary metabolites of styrene, mandelic acid (MA) and phenylglyoxylic acid (PGA), among workers exposed to styrene in the reinforced-plastics industry and in unexposed subjects. We also wished to determine whether cigarette smoking influenced adduct levels among these subjects. METHODS: A group of 22 male workers was selected on basis of an expectedly high level of exposure to styrene, and a group of 15 controls was selected from hospital blood donors and hospital staff. In the exposed group, MA and PGA were quantified by high-performance liquid chromatography (HPLC) analysis of urine samples collected prior to the work shift. The SO adducts were cleaved from cysteine residues by reaction with Raney nickel to give 1-phenylethanol (1-PE) and 2-phenylethanol (2-PE), which, after derivatization, were measured using gas chromatography-mass spectrometry (GC-MS) in the negative-chemical-ionization (NCI) mode. RESULTS: The estimated mean levels of MA and MA + PGA were 74 and 159 mg/g creatinine, respectively. Using the levels of urinary metabolites, an average styrene concentration of about 100 mg/m3 in the workplace air was estimated. The mean levels of 2-PE and 1-PE adducts in exposed workers were 2.84 and 0.60 nmol/g Alb and 5.44 and 0.43 nmol/g Hb, respectively. When subjects were stratified by level of urinary metabolites [zero (controls), low-level exposure (MA + PGA < or = 159 mg/g creatinine), and high-level exposure (MA + PGA > 159 mg/g creatinine)] and smoking status (smokers versus nonsmokers), a difference in Alb adduct levels was found among the groups (2-PE P = 0.002, I-PE P = 0.052). The difference in 2-PE-Alb levels was related to exposure category, to smoking status, and to their interaction. Correlations at or near a 0.05 level of significance were observed among the workers (n = 22) between individual levels of SO-protein adducts and MA + PGA (2-PE Alb, r = 0.54, 2-PE Hb, r = 0.40). CONCLUSION: Our data suggest that only exposure to relatively high levels of styrene allows a clear relationship to be detected between styrene exposure and SO adducts, due in part to the effects of cigarette consumption and to the high background levels of these adducts observed in unexposed subjects.

Comparison of styrene-7,8-oxide adducts formed via reaction with cysteine, N-terminal valine and carboxylic acid residues in human, mouse and rat hemoglobin.

The reactive metabolite of styrene, styrene-7,8-oxide (SO), reacts with a variety of nucleophilic sites in hemoglobin (Hb) to form SO-Hb adducts. Following the in vitro incubation of SO with blood from humans, NMRI mice and Sprague-Dawley rats, the second-order reaction rate constants were determined for the reaction of SO with cysteine (through both the alpha- and beta-carbons of SO), N-terminal valine (through the beta-carbon of SO), and carboxylic acid (presumably through both the alpha- and beta-carbons of SO) residues in Hb. The rate constants for cysteine adducts vary dramatically between species [2.04, 10.7, 133 L (mol Hb)-1 h-1 (alpha binding) for humans, mice and rats, respectively] and [0.078, 2.16, 20.4 L (mol Hb)-1 h-1 (beta binding), respectively]. The considerably higher rate of reaction with cysteine in rat Hb probably reflects the presence of an additional cysteine residue at position beta 125. Although the rate constants for valine adducts (1.82, 0.80, 0.29 L (mol Hb)-1 h-1, respectively) and COOH adducts (3.55, 1.94, 2.37 L (mol Hb)-1 h-1, respectively) are much more consistent, the inter-species differences are statistically significant for the reaction of SO with the N-terminal valine of Hb. Following the i.p. administration of styrene to mice and styrene and SO to rats, the levels of adducts at each of these sites were used in conjunction with the calculated rate constants to predict the integrated blood doses of SO. While the SO doses predicted from cysteine and valine adducts were very similar, that based upon COOH-binding was significantly different, presumably due to the instability of SO-COOH adducts. This research affirms the use of both cysteine and valine adducts, but not carboxylic acid adducts, as biomarkers of exposure to styrene and SO.

Measurement of benzene oxide in the blood of rats following administration of benzene.

Although it is generally assumed that metabolism of benzene proceeds through an initial step involving oxidation to benzene oxide (BO) by CYP450 in the liver, the production of BO has never been unambiguously confirmed in animals dosed with benzene. Furthermore, prevailing hypotheses of the mechanism by which benzene causes cancer have ignored the possibility that BO might play a direct role, despite the fact that BO is electrophilic, binds covalently to cell macromolecules and is presumably genotoxic. A likely reason for this lack of attention to the role of BO in the carcinogenesis of benzene is the presumption that this epoxide is too reactive to escape the hepatocyte after it is formed. We employed gas chromatography-mass spectrometry to measure BO in the blood of F344 rats, both in vitro and up to 24 h following oral administration of benzene. Surprisingly, BO was relatively stable in rat blood at 37 degrees C (estimated half-life = 7.9 min) and, after administering a single dosage of 400 mg benzene/kg body wt, a blood concentration of 90 nM BO (8.5 ng/ml) was measured for approximately 9 h. Using a published PBPK model we estimate that approximately 4.3% of the metabolized dose of benzene was released as BO from the liver into blood. This confirms that BO is, indeed, formed from metabolism of benzene and is sufficiently stable to be distributed throughout the body at levels which are likely to be greater than those of the other electrophilic benzene metabolites.

An investigation of multiple biomarkers among workers exposed to styrene and styrene-7,8-oxide.

Investigations of cancer and cytogenetic damage among reinforced-plastics workers have produced contradictory results. In all studies, the focus has been on styrene rather than the carcinogen, styrene-7,8-oxide (SO), traces of which are generated during the manufacturing process. Because styrene is present at very high levels and is metabolized almost exclusively through SO, coexposures to SO have been discounted. This study investigated the relative contributions of airborne styrene and SO and of smoking toward several SO-specific biomarkers (DNA and albumin adducts) and sister chromatid exchanges in the blood of 48 reinforced-plastics workers. Among individual subjects, albumin and DNA adducts as well as sister chromatid exchanges were significantly correlated with styrene exposure. However, among the 20 subjects with measurements to both styrene and SO, albumin adducts were significantly correlated with exposure to SO but not to styrene. Finally, among the 10 job groups, surprisingly strong correlations (0.709 < or = r < or = 0.966) were found between all SO biomarkers and exposure to SO but not to styrene. Calculations suggest that SO was about 2000 times more effective than styrene in producing SO biomarkers. After accounting for the disparate exposures to the two chemicals, a typical worker received 71% of the systemic dose of SO via inhalation; nonetheless, 5 of the 20 subjects received the majority of the SO dose from styrene. Cigarette smoking increased levels of SO-albumin and SO-DNA adducts, suggesting that SO was a constituent of tobacco smoke. We conclude that inhalation of SO should be considered in any interventions to reduce health risks.

The use of protein adducts to investigate the disposition of reactive metabolites of benzene.

Benzene is metabolized to a number of electrophilic species that are capable of binding to both DNA and proteins. We used adducts of hemoglobin (Hb) and bone marrow proteins to study the disposition of three benzene and metabolites (benzene oxide [BO], 1,2-benzoquinone [1,2-BQ], and 1,4-benzoquinone [1,4-BQ]) in F344 rats and B6C3F1 mice following a single oral dosage of [13C6]benzene and/or [14C]benzene. Our assays focused upon cysteine adducts that accounted for 38 to 45% of protein binding to Hb and 63 to 81% of protein binding to bone marrow. Although both mice and rats showed dose-related increases in Hb and bone marrow protein adducts of BO and of the two benzoquinones, large intertissue and interspecies differences were noted, suggesting different preferences in metabolic pathways. The highest levels of adducts in mice were of 1,4-BQ (10-27% of all cysteine adducts), while in rats, BO adducts predominated in Hb (73% of all cysteine adducts) and 1,2-BQ adducts predominated in the bone marrow (14% of all cysteine adducts). High background levels of 1,2-BQ and 1,4-BQ adducts were also detected in both species, indicating that the toxic effects of quinone metabolites may only be important at high levels of benzene exposure.

Analysis of hemoglobin adducts of benzene oxide by gas chromatography-mass spectrometry.

A method is reported for measuring cysteinyl adducts of the benzene metabolite benzene oxide (BO) with hemoglobin (Hb) in blood from humans or rodents exposed to benzene. After reacting the purified, dried protein with trifluoroacetic anhydride and methanesulfonic acid, the resulting phenyltrifluorothioacetate is extracted, washed, and detected by GC-MS in the negative-ion chemical ionization mode. The analysis of Hb adducts of BO from rats dosed with 50-400 mg/kg [13C6]benzene via this method resulted in values which were generally consistent, though slightly lower, than those obtained using an established method. However, while 3 weeks were required to process the samples by the former method, only 2 days were needed with the new procedure. This new method should, therefore, prove to be reliable and convenient for the rapid quantitation of cysteine-bound Hb adducts of BO.

Determination of albumin and hemoglobin adducts in workers exposed to styrene and styrene oxide.

Hemoglobin and albumin adducts of the carcinogen styrene-7,8-oxide (SO) were measured in 48 workers exposed to both styrene and SO in a boat manufacturing plant. Personal exposures to both substances were measured repeatedly over the course of 1 year (styrene:0.9-235 mg/m3 with a mean of 64.3 mg/m3 for 48 subjects; S0: 13.4-525 mu g/m3 with a mean of 159 mu g/m3 for 20 subjects). Cysteine and carboxylic acid adducts of SO with hemoglobin and albumin were assayed on one or more occasions for each subject. The proteins were subjected to base hydrolysis to release styrene glycol, representing carboxylic acid-bound SO, and were then treated with Raney nickel to release 1-phenylethanol and 2-phenylethanol, representing cysteine-bound SO. These three analytes were extracted, derivatized, and analyzed by gas chromatography-mass spectrometry. No evidence was found of any exposure-related increase in hemoglobin adducts. In contrast, albumin adducts were found to increase with exposures to either styrene or SO, the latter apparently being more important. This suggests that exposure to low levels of SO in the air may be important among workers in the reinforced plastics industry. Significant levels of SO adducts of albumin and hemoglobin were also detected in proteins obtained from persons without occupational exposure to styrene or to SO. This finding opens the possibility that SO is either a dietary or an environmental contaminant or is produced endogenously.

Comparison of protein adducts of benzene oxide and benzoquinone in the blood and bone marrow of rats and mice exposed to [14C/13C6]benzene.

Protein binding of hemoglobin (Hb) and bone marrow was used to compare in vivo reactions of 3 electrophilic metabolites of benzene, i.e., benzene oxide and 1,2- and 1,4-benzoquinone (1, 2-BQ and 1, 4-BQ), in F344 rats and B6C3F1 mice. Following a single p.o. administration of a mixture of [14C]- and [13C6]benzene between 50 and 400 mg/kg body weight, cysteine adducts of benzene oxide, 1,2-BQ, and 1,4-BQ were assayed, and the proportions of cysteine-bound adducts to total protein binding were estimated. Although dose-related production of each adduct was seen, large differences were observed between species and tissues. With rat Hb, benzene oxide adducts represented 27% of the total Hb binding and 73% of the cysteinyl binding, whereas quinone adducts represented relatively small proportions. However, with mouse Hb, the 1,4-BQ adducts accounted for 5.5% of the total Hb binding and 12.2% of the cysteinyl binding, while 1,2-BQ and benzene oxide each accounted for less than 3% of the total. In the bone marrow of both rats and mice, BQ adducts were more abundant than those of benzene oxide. However, adducts of 1,2-BQ predominated in rat marrow (9% of binding), whereas adducts of 1,4-BQ were more abundant in the mouse (21% of binding). The average blood concentrations of 1,4-BQ were estimated from the adduct levels and reaction-rate constants to be 2-5-fold higher in the mouse than in the rat. This work suggests that BQ binding is favored over that of benzene oxide in the bone marrow; however, high background levels of BQ adducts, observed with Hb and bone marrow proteins, suggest that any toxic effects of the quinones should only arise from high exposures to benzene.

Application of Raney nickel to measure adducts of styrene oxide with hemoglobin and albumin.

Adducts of styrene 7,8-oxide (SO) with the blood proteins, Hb and albumin (Alb), were measured following treatment of the proteins with Raney nickel (Ra-Ni) to cleave 2-phenylethanol, which was subsequently assayed. Internal standards were prepared by modifying Hb and Alb with 4-methyl-SO to produce similar adducts. In a preliminary experiment with human blood, which had been modified with [14C]SO in vitro, it was determined that Ra-Ni released 6% of the total binding to globin and 76% of the total binding to Alb. Since Ra-Ni primarily cleaves covalently-bound sulfur species, this suggests that much more binding to human serum Alb occurred at a free cysteine residue than to human Hb. The overall rate of reaction of SO at 37 degrees C in vitro had a half-time of 0.70 h in human blood and of 0.36 h in blood from Sprague-Dawley rats. Adducts of SO with Hb and Alb were measured following SO modification of blood from humans and rats in vitro at 37 degrees C. Slopes of the linear relationships between adduct level and SO concentration allowed the following second-order rate constants [(L/mol of protein)/h] to be estimated: rat Hb, 72; human Hb, 2.4; rat Alb, 63; human Alb, 32. These rate constants show that intrinsic reactivity of SO toward the cysteine residue of human Hb is much lower than that of rat Hb or of Alb from either species. Adducts of Hb and Alb were also measured following administration of both SO and styrene to rats.(ABSTRACT TRUNCATED AT 250 WORDS)