Hepatic Cytochrome P450 Isozymes Research Articles

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.

Purification and characterization of the dog hepatic cytochrome P-450 isozyme responsible for the metabolism of 2,2′,4,4′,5,5′-hexachlorobiphenyl

The biochemical basis for the marked difference in the rate of the hepatic metabolism of 2,2′,4,4′,5,5′-hexachlorobiphenyl (245-HCB) by Beagle dogs and Sprague—Dawley rats has been investigated. Control dog liver microsomes metabolize this substrate 15 times faster than control rat liver microsomes. Upon treatment with phenobarbital (PB), at least two cytochrome P-450 isozymes are induced in the dog, and the hepatic microsomal metabolism of 245-HCB is increased on both a per nanomole P-450 basis (twofold) and a per milligram protein basis (fivefold). One of the PB-induced isozymes, PBD-2, has been purified to a specific content of 17–19 nmol/mg protein and to >95% homogeneity, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a reconstituted system containing cytochrome b 5, this isozyme shows an activity toward 245-HCB which is greater than threefold that seen in intact liver microsomes from PB-induced dogs. A reconstituted system containing the major isozyme induced by PB in the rat (PB-B) metabolizes 245-HCB at 1 10 the rate observed with purified PBD-2. Antibody inhibition studies have shown that PBD-2 accounts for >90% of the hepatic microsomal metabolism of 245-HCB in control and PB-induced dogs, while PB-B only accounts for about half of the metabolism of this compound by microsomes obtained from PB-treated rats. Immunoblot analysis has revealed that the level of PBD-2 in dog liver microsomes increases nearly sixfold with PB treatment, and this increase correlates well with the fivefold increase in the rate of hepatic microsomal metabolism of 245-HCB by dogs. Together these data support a primary role for isozyme PBD-2 in the hepatic metabolism of 245-HCB in control and PB-induced dogs. In addition, these results suggest that, in contrast to rats, dogs can readily metabolize 245-HCB as a result of the presence of a cytochrome P-450 isozyme with efficient 245-HCB metabolizing activity.

Relationship between paracetamol binding to and its oxidation by two cytochromes P-450 isozymes--a proton nuclear magnetic resonance and spectrophotometric study.

From the hepatic cytochrome P-450 isozymes b and c isolated from rats treated with phenobarbital and 3-methylcholanthrene respectively, only cytochrome P-450c was found to be active in the oxidation of paracetamol, in the presence of glutathione ultimately leading to the formation of the 3-glutathionyl conjugate. Paracetamol interacted with both cytochrome P-450b and c, as shown by difference spectrophotometry. Cytochrome P-450b was found to have a higher affinity for paracetamol than cytochrome P-450c and demonstrated a type I spectral change, whereas in the case of cytochrome P-450c a reverse type I spectral change was observed. Proton n.m.r. longitudinal relaxation rate measurements revealed that in the case of cytochrome P-450c, paracetamol was orientated with its phenolic hydroxyl group in closest proximity to the central haem iron ion. In the case of cytochrome P-450b, the acetylamino group of paracetamol most closely approached the haem iron ion.

Use of monoclonal antibodies to characterize the induction response of the cytochrome P-450-dependent mixed function oxidase system to nitrofluoranthenes.

In prior studies with neonatal rats we have suggested that nitrated polycyclic aromatic hydrocarbons (NPAH) are 3-methylcholanthrene (3-MC) type of inducers of cytochrome P-450. These observations have been extended by studying the effect of fluoranthene (FL) and its nitrated derivative, 3-nitrofluoranthene (3-NF) and a mixture of nitrated fluoranthenes (NFs) on the induction of hepatic and pulmonary monooxygenase activities in adult rats. We have characterized the effect of these compounds on hepatic cytochrome P-450 isozyme(s) using immunoblot analysis. The administration of 3-NF and NFs to rats resulted in highly significant induction (1.9- to 5.8-fold) of hepatic and pulmonary aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin-O-deethylase (ERD) activities. FL was either ineffective or much less effective in inducing these enzyme activities. The enzyme induction response to these compounds occurred in the following order: NFs greater than 3-NF greater than FL. SDS-PAGE of hepatic microsomes prepared from FL-, 3-NF- and NFs-treated animals revealed a higher content of protein migrating in the P-450 region. Characterization of isozymes of P-450 was carried out by Western blot analysis with highly specific monoclonal antibodies (MAb) raised against 3-MC-specific P-450 (MAb 1-7-1) and phenobarbital-specific P-450 (MAb-2-66-3) isozymes. Hepatic microsomes prepared from 3-NF- and NFs-treated rats showed two distinct immunoprecipitin bands with MAb 1-7-1 whereas microsomes prepared from FL-treated animals showed a sharp band with MAb 2-66-3. MAb 1-7-1 significantly inhibited (approximately 80%) AHH activity induced by 3-NF and NFs. On the other hand FL-induced AHH activity was only moderately (approximately 30%) inhibited by MAb 1-7-1 whereas higher inhibition (approximately 60%) was observed with MAb 2-66-3. Analysis of BP metabolites by h.p.l.c. revealed enhanced production of metabolites by liver microsomes from 3-NF- and NFs-treated animals. The formation of BP 7,8-diol was 1.8- to 2.4-fold increased following treatment of animals with 3-NF and NFs respectively. Addition of MAb 1-7-1 to a microsomal mixture from 3-NF- and NFs-treated rats inhibited the formation of BP phenols (60-75%) and BP 7,8 diol (52-60%). These inhibitory effects were not observed with microsomes prepared from FL-treated rats. These studies suggest that NFs induce specific monooxygenases in liver and that they are inducers of P-450 isozymes c and d.

Mechanism-based in vivo inactivation of lauric acid hydroxylases.

The hepatic cytochrome P-450 isozymes that catalyze omega- and (omega - 1)-hydroxylation of lauric acid are specifically inactivated in vitro but not in vivo by 10-undecynoic acid. The lack of in vivo activity may result from rapid degradation of the inhibitor by beta-oxidation. Strategies for the construction of fatty acid analogues that retain the ability to inactivate fatty acid hydroxylases but are resistant to metabolic degradation have therefore been sought. Fatty acid analogues in which the carboxylic acid group is replaced by a sulfate moiety, or in which two methyl groups are placed vicinal to the carboxylic acid group, have been found to inactivate lauric acid hydroxylases in vitro and in vivo without causing time-dependent inhibition of ethoxycoumarin O-deethylation or N-methyl-p-chloroaniline N-demethylation.

N-demethylation of N-nitrosodimethylamine catalyzed by purified rat hepatic microsomal cytochrome P-450: Isozyme specificity and role of cytochrome b5

Metabolism of the potent hepatocarcinogen N-nitrosodimethylamine (NDMA) was evaluated in reconstituted monooxygenase systems containing each of 11 purified rat hepatic cytochrome P-450 isozymes. The reaction has an absolute requirement for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH, as well as a partial dependence on dilauroylphosphatidylcholine. Of the cytochrome P-450 isozymes evaluated, only cytochrome P-450j, purified from livers of ethanol- or isoniazid-treated rats, had high catalytic activity for the N-demethylation of NDMA. At substrate concentrations of 0.5 and 5 m m, rates of NDMA metabolism to formaldehyde catalyzed by cytochrome P-450j were at least 15-fold greater than the rates obtained with any of the other purified isozymes. At the pH optimum (~6.7) for the reaction, the K m,app and V max were 3.5 m m and 23.9 nmol/min/nmol cytochrome P-450j, respectively. With hepatic microsomes from ethanol-treated rats, which contain induced levels of cytochrome P-450j, the K m,app and V max were 0.35 m m and 3.9 nmol/min/nmol cytochrome P-450, respectively. Inclusion of purified cytochrome b 5 in the reconstituted system containing cytochrome P-450j caused a six-fold decrease in K m,app (0.56 m m) of NDMA demethylation with little or no change in V max (29.9 nmol/min/nmol cytochrome P-450j). Trypsinsolubilized cytochrome b 5, bovine serum albumin, or hemoglobin had no effect on the kinetic parameters of the reconstituted system, indicating a specific effect of intact cytochrome b 5 on the K m,app of the reaction. These results demonstrate high isozyme specificity in the metabolism of NDMA to an ultimate carcinogen and further suggest an important role for cytochrome b 5 in this biotransformation process.

Dissociation of increased lauric acid ω-hydroxylase activity from the antilipidemic action of clofibrate

Clofibrate, an antilipidemic drug that acts by a still obscure mechanism, is known to specifically increase up to 30-fold the activity of the hepatic cytochrome P-450 isozyme that ω-hydroxlates lauric acid. The thesis that accelerated catabolism of medium-length fatty acids initiated by ω-hydroxylation contributes significantly to the antilipidemic action of clofibrate has been examined by measuring the impact on serum triglyceride levels of coadministering clofibrate and a suicide substrate that inactivates the hydroxylase. The results suggest that the antilipidemic action of clofibrate does not depend critically on the enhanced ω-hydroxylation of fatty acids.

ω-1 and ω-2 hydroxylation of prostaglandins by rabbit hepatic microsomal cytochrome P-450 isozyme 6

Incubation of prostaglandin E 1 (PGE 1) with liver microsomes from control rabbits and from rabbits treated with ethanol or imidazole yielded 18-, 19-, and 20-hydroxy metabolites, representing hydroxylation at ω-2, ω-1, and ω carbons, respectively. The current investigation demonstrates that rabbit liver P-450 isozyme 6 effectively catalyzes the ω-1 andg w-2 hydroxylation of PGE 1 and PGE 2. Additionally, a small amount of product with Chromatographic characteristics of the corresponding 20-hydroxy metabolite has been detected. The incorporation of cytochrome b 5 into the reconstituted system did not enhance the rate of PGE 1 hydroxylation and had no effect on the ratio of products formed. The K m value for the ω-1 and ω-2 hydroxylation of PGE 1 with P-450 isozyme 6 from imidazole-treated rabbits was approximately 140 μ m; the V max's (nmol product min −1 nmol P-450 −1) were 2.1 and 1.1 for the ω-1 and ω-2 hydroxylations, respectively. These rates represent the highest activities by hepatic P-450 isozymes for hydroxylation of PGs, and suggest that isozyme 6 is responsible for the ω-2 hydroxylation of PGEs observed in rabbit liver microsomes.

Effects of the induction of hepatic microsomal metabolism on the toxicity of cyclophosphamide.

Cyclophosphamide (CP) administration to rats in a single i.p. dose (200 mg kg-1), while producing urinary bladder toxicity and 30-40% depression of the hepatic microsomal mixed function oxidase (MFO), failed to produce any depression of MFO activities in extrahepatic tissues such as lung, kidney and intestine. Phenobarbital pretreatment of the rats, which is known to enhance hepatic microsomal activation of CP, protected against CP-induced urinary bladder toxicity and the depression of hepatic MFO activities. This protection appears to be, at least in part, related to phenobarbital induction of hepatic cytochrome P-450 isozyme(s) that metabolizes CP to a new metabolite tentatively identified as didechlorodihydroxycyclophosphamide.

NH 2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450 1

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15–20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65–87% hydrophobic amino acids, and only one charged amino acid (Asp) in the ammo-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11–12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes ( P-450a-e).

Rat hepatic cytochrome P-450 isozyme specificity for the metabolism of the steroid sulfate, 5α-androstane-3α,17β-diol-3,17-disulfate

Nine rat hepatic microsomal cytochromes P-450 ( P-450a- P-450i) have been purified to electrophoretic homogeneity and assayed as potential catalysts of 15β-hydroxylation of 5α-androstane-3α,17β-diol-3,17-disulfate in a reconstituted system containing NADPH-cytochrome c reductase and dilauroylphosphatidylcholine. Of the nine isozymes, only cytochrome P-450i, which is present in adult female but not adult male rats, catalyzes the hydroxylation of 5α-androstane-3α,17β-diol-3,17-disulfate. The reaction has an absolute requirement for cytochrome P-450i, NADPH-cytochrome c reductase, and NADPH, as well as a marked dependence on dilauroylphosphatidylcholine. Under optimal conditions, the K m,app was 69 μ m, and the V maxwas 7.8 nmol min −1 nmol cytochrome P-450i −1. The affinity of cytochrome P-450i for the substrate, as expressed by the apparent spectral dissociation constant ( K sapp ), was 20 μ m. This female-specific isozyme had very low catalytic activity toward testosterone, and the metabolite profile from testosterone was distinct compared to the profiles obtained with the other eight isozymes. The results with androstane disulfate indicated that cytochrome P-450i is responsible for the sex-specific microsomal 15β-hydroxylase system in adult female rat liver, originally described by Gustafsson and Ingelman-Sundberg [(1974) J. Biol. Chem. 249: 1940–1945].

Regio- and stereoselective metabolism of two C19 steroids by five highly purified and reconstituted rat hepatic cytochrome P-450 isozymes.

High pressure liquid chromatographic systems capable of resolving at least 28 known and potential metabolites of 17 beta-hydroxy-4-androsten-3-one (testosterone) and 4-androstene-3,17-dione (androstenedione) were used to quantitatively assess the metabolism of the two steroids in monooxygenase systems reconstituted with five purified rat liver cytochrome P-450 isozymes. Cytochromes P-450a, -b, -c, -d, and -e catalyzed the oxidation of testosterone at overall rates of 21, 27, 2, 0.7, and 3 nmol/min/nmol of cytochrome P-450, respectively; while the corresponding rates for total androstenedione metabolism were 12, 62, 1.5, 0.3, and 5. Cytochrome P-450a catalyzed the oxidation of testosterone and androstenedione almost exclusively to their respective 7 alpha-hydroxy metabolites. Cytochrome P-450b catalyzed the oxidation of testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone in approximately equal molar ratios. However, this same hemoprotein exhibited a marked stereoselectivity in the metabolism of androstenedione since the molar ratio of 16 alpha- and 16 beta-hydroxyandrostenedione was greater than 1:10. Cytochrome P-450e catalyzed the oxidation of both steroids to the same products as cytochrome P-450b, but at approximately 10% of the rate. Cytochromes P-450c and P-450d catalyzed the oxidation of testosterone and androstenedione regio- and stereospecifically to their respective 6 beta-hydroxy metabolites. These results indicate that certain cytochrome P-450 isozymes show marked positional specificity in the metabolism of both testosterone and androstenedione, and that the rate as well as stereoselectivity of the oxidative reactions can be markedly dependent on subtle differences in the structure of the steroid substrate.

On the glycosylation state of five rat hepatic microsomal cytochrome P-450 isozymes.

The glycosylation states of five rat hepatic microsomal cytochrome P-450 isozymes (cytochromes P-450a, P-450b, P-450c, P-450d, and P-450e) were examined by quantitative carbohydrate analysis. Carbohydrate content of the purified enzymes as determined by acid hydrolysis, reduction, and gas chromatography of the alditol acetates revealed only trace amounts of neutral and amino hexoses in each of the five isozymes. Levels of mannose ranged from 0.3 to 1.7 mol/mol of cytochrome P-450 whereas levels of galactose were less than or equal to 0.2 mol/mol of cytochrome P-450 for the five hemoproteins. The amino sugars glucosamine and galactosamine were usually present at levels less than or equal to 0.2 mol/mol of cytochrome P-450, although one preparation of cytochrome P-450b had as much as 0.5 mol of glucosamine/mol of cytochrome P-450. Other carbohydrate residues (xylose and arabinose) were not detected in significant quantities. Since N- and O-glycosylation of proteins occurs primarily through N-acetylglucosaminyl and N-acetylgalactosaminyl residues, respectively, the lack of significant amounts of these amino sugars indicates that these five cytochrome P-450 isozymes are not normally glycosylated in the native state. Purified NADPH-cytochrome c reductase, which functions as an electron donor for microsomal cytochrome P-450, contained no detectable quantities of hexose sugars.

Purification, characterization and regulation of five rat hepatic microsomal cytochrome P-450 isozymes.

1. Five hepatic microsomal cytochrome P-450 isozymes (cytochromes P-450a, P-450b, P-450c, P-450d, P-450e) have been purified to apparent homogeneity from immature male rats treated with various xenobiotics. 2. The unique electrophoretic properties, substrate specificities and spectral characteristics of these haemoproteins have been compared and contrasted. 3. Structural studies of these cytochrome P-450 isozymes have included NH2-terminal amino acid sequence analyses, as well as electrophoretic profiles of limited proteolytic digests and cyanogen bromide fragments of the haemoproteins. 4. Specific antibodies have been prepared against four of the isozymes and used to evaluate immunochemical relationships among these cytochrome P-450s by Ouchterlony double-diffusion analyses. 5. The levels of some of these cytochrome P-450 isozymes have been quantified immunologically in hepatic microsomal preparations from untreated rats and following induction by phenobarbital, 3-methylcholanthrene or Aroclor 1254. 6. Antibodies directed against cytochromes P-450a and P-450b have been used to establish the presence of more than one 7 alpha- and 16 alpha-testosterone hydroxylase in rat hepatic microsomes. The relative contributions of cytochromes P-450c and P-450d to the overall microsomal metabolism of benzo[a]pyrene have been evaluated using antibodies to these haemoproteins.

Food Exposures to Polychlorinated Biphenyls

ArticlesOpen AccessFood Exposures to Polychlorinated Biphenyls Albert C. Kolbye Jr. Albert C. Kolbye Jr. Search for more papers by this author Published:1 April 1972https://doi.org/10.1289/ehp.720185Cited by:21AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit FiguresReferencesRelatedDetailsCited By ANDERSON H (1989) General population exposure to environmental concentrations of halogenated biphenyls Halogenated Biphenyls, Terphenyls, Naphthalenes, Dibenzodioxins and Related Products, 10.1016/B978-0-444-81029-8.50015-1, (325-344), . Duignan D, Sipes I, Leonard T and Halpert J (1987) Purification and characterization of the dog hepatic cytochrome P-450 isozyme responsible for the metabolism of 2,2′,4,4′,5,5′-hexachlorobiphenyl, Archives of Biochemistry and Biophysics, 10.1016/0003-9861(87)90396-1, 255:2, (290-303), Online publication date: 1-Jun-1987. 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Peterson R, Seymour J and Allen J (1976) Distribution and biliary excretion of polychlorinated biphenyls in rats, Toxicology and Applied Pharmacology, 10.1016/0041-008X(76)90191-5, 38:3, (609-619), Online publication date: 1-Dec-1976. Khan M, Rao R, Novak A and Murthy M (2009) Polychlorinated biphenyls (Pcbs) in food, C R C Critical Reviews in Food Science and Nutrition, 10.1080/10408397609527204, 7:2, (103-145), Online publication date: 1-Jan-1976. Greb W, Klein W, Coulston F, Golberg L and Korte F (2013) Beiträge zur Ökologischen Chemie LXXXIII† In Vitro Metabolism of Polychlorinated Biphenyls-14C, Bulletin of Environmental Contamination and Toxicology, 10.1007/BF01721847, 13:4, (424-432), Online publication date: 1-Apr-1975. Fries G and Marrow G (1975) Excretion of Polybrominated Biphenyls Into the Milk of Cows, Journal of Dairy Science, 10.3168/jds.S0022-0302(75)84661-3, 58:6, (947-951), Online publication date: 1-Jun-1975. Mehlman M, Yin L and Nielsen R (1974) Metabolic changes in “frozen-clamped” livers of rats caused by ingestion of polychlorinated biphenyls, Toxicology and Applied Pharmacology, 10.1016/0041-008X(74)90201-4, 27:2, (300-307), Online publication date: 1-Feb-1974. Litterst C, Mimnaugh E, Edelman S, Peters E, Miller E and Van Loon E (1974) Distribution and biochemical effects of pentylenetetrazol in brains of swine treated with a polychlorinated biphenyl, Chemico-Biological Interactions, 10.1016/0009-2797(74)90030-1, 8:4, (217-223), Online publication date: 1-Apr-1974. Willett L (1974) Coatings as Barriers to Prevent Polychlorinated Biphenyl Contamination of Silage, Journal of Dairy Science, 10.3168/jds.S0022-0302(74)84970-2, 57:7, (816-825), Online publication date: 1-Jul-1974. Bruckner J, Khanna K and Cornish H (1974) Effect of prolonged ingestion of polychlorinated biphenyls on the rat, Food and Cosmetics Toxicology, 10.1016/0015-6264(74)90004-2, 12:3, (323-330), Online publication date: 1-Jun-1974. Kimbrough R, Barnes M and Greig J (2008) The Toxicity of Polychlorinated Polycyclic Compounds and Related Chemicals, CRC Critical Reviews in Toxicology, 10.3109/10408447309025705, 2:4, (445-498), Online publication date: 1-Jan-1973. (1972) Preface, Environmental Research, 10.1016/0013-9351(72)90002-3, 5:3, (253), Online publication date: 1-Sep-1972. Gramaglia C and Babut M (2014) L’expertise à l’épreuve d’une controverse environnementale et sanitaire : la production des savoirs et des ignorances à propos des PCB du Rhône (France), VertigO, 10.4000/vertigo.15067:Volume 14 Numéro 2 Vol. 1April 1972Metrics About Article Metrics Publication History Originally published1 April 1972Published in print1 April 1972 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. 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