Reflections on the development of a host-response diagnostic test for periodontal disease.

This manuscript reviews the sequence of events surrounding the development of a proposed diagnostic test for periodontal disease based on analysis of a host mediator in gingival crevicular fluid (GCF). The mediator is lysosomal beta-glucuronidase (beta G), a marker of granule release from polymorphonuclear leukocytes. Preliminary studies were conducted to evaluate the most reproducible method of collecting GCF and analyzing the data. This was followed by analysis of the mediator in GCF in a variety of cross-sectional and short-term longitudinal studies, and eventually by a study of the relationship of beta G in GCF to disease progression (probing attachment loss) in a single center longitudinal trial. Development of an academic-industrial collaboration allowed the project to progress, as a rapid, practical assay system was developed, and a multi-center longitudinal trial was conducted. The multicenter trial indicated that patients with elevated beta G in GCF were at 6 to 14 times higher risk for experiencing probing attachment loss. The technical, regulatory, business and marketing issues surrounding the introduction of a diagnostic test for periodontal disease were considered. While technical and regulatory issues were not major impediments, business and marketing concerns contributed to a decision to halt development of the test. Specifically, there was concern about acceptance of the test by practitioners and third party reimbursement. Underlying these issues were the multifactorial nature of periodontal disease, and the lack of a direct cause and effect relationship of test results to disease outcome. While analysis of beta G in GCF is not currently available as a diagnostic test for periodontal disease, the development process has identified important issues for the introduction of diagnostic tests, including consideration of the relationship of test results to treatment, the need to integrate diagnostic tests into dental practice, and an appreciation of the financial and health care benefits derived by practitioners and patients with use of such tests.

Specific activity of polypyrrole nanoparticulate immunoreagents: comparison of surface chemistry and immobilization options.

Polypyrrole-based colloids with differing surface chemistries were compared with respect to the specific activity of immobilized antibody. Monoclonal antibody to the alpha subunit of human chorionic gonadotropin (hCG) was modified by incorporation of cystamine into the Fc-carbohydrate, followed by reduction with dithiothreitol resulting in the generation of 4.5 free thiols per IgG. The reduced IgG was added to clean, unmodified and surface-modified polypyrrole colloids. Functionalized colloids included carboxylate-modified polypyrrole, poly[pyrrole-co-1-(2-carboxyethyl) pyrrole]-silica composite, and amine forms of the carboxylated colloids. The amine-functionalized colloids were subsequently treated with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate to provide thiol-reactive maleimide surface groups. Following the conjugation of IgG to the colloids, bound and soluble antibody activity was quantitated using a sequentially competitive immunoassay for hCG, based on an automated commercial hCG kit. The results indicated that all forms of polypyrrole retained the equivalence of between 12 and 33 micrograms of IgG activity/mg of colloidal solids, relative to the unmodified soluble IgG.

The relationship of beta-glucuronidase activity in crevicular fluid to probing attachment loss in patients with adult periodontitis. Findings from a multicenter study.

Analysis of gingival crevicular fluid (GCF) offers a non-invasive means of studying the host response in periodontal disease, and may provide an early indication of the patient at risk for active periodontitis. A number of host markers have been studied for their relationship to disease activity (probing attachment loss or PAL). GCF levels of the acid glycohydrolase beta-glucuronidase (beta G), a marker of primary granule release from polymorphonuclear leukocytes (PMN), have been shown to identify patients with periodontitis at risk for additional PAL. In this multicenter trial, we evaluated (a) the short-term effect of conservative periodontal therapy on beta G activity in GCF, and (b) the relationship of persistently elevated beta G activity to PAL in patients who were monitored for 6 months. The study population included a total of 140 patients with chronic adult periodontitis. 130 patients were on a regular recall schedule, and 10 were previously untreated. After collection of baseline clinical data at all sites, and analysis of beta G in GCF from one site (mesiobuccal) per tooth, the patients received a scaling and prophylaxis. Two weeks later patients were seen for collection of GCF. If elevated enzyme activity was found at 2 weeks, the patients were seen at 3 months for a clinical exam and GCF collection. Clinical parameters were collected from all patients at 6 months. Therapy tended to reduce beta G activity in GCF.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship of beta-glucuronidase activity in crevicular fluid to clinical parameters of periodontal disease. Findings from a multicenter study.

Previous reports have suggested that persistently elevated levels of the acidic glycohydrolase beta-glucuronidase (beta G) in gingival crevicular fluid (GCF) can identify patients with chronic adult periodontitis who are at risk for future probing attachment loss (PAL). To comprehensively study beta G activity in GCF, a multicenter trial examining the relationship of the enzyme in GCF to traditional clinical parameters of periodontal disease and PAL was conducted. In this report, the baseline data was used to evaluate the relationship of beta G activity in GCF to traditional parameters of periodontal disease. The study group included 130 patients who had been treated for periodontal disease and were on a regular recall schedule, and 10 patients with chronic adult periodontitis who had never received treatment. Upon entering the longitudinal trial, the patients were examined, and a standardized 30-s GCF sample was collected from the mesiobuccal crevice of all study teeth. As a control, GCF samples and clinical data were collected from 62 patients with a healthy periodontium or mild inflammatory gingivitis without loss of probing attachment. At baseline, beta G activity for the periodontitis patients ranged from 0 to 1704 Units (U), with a median of 32 U. beta G could not be detected in 0.2% of samples (activity < or = 2.0 U). The 90% cumulative relative frequency was 139 U. For the healthy/gingivitis subjects beta G activity ranged from 0 to 504 U, with a median of 22 U. Enzyme was not detectable in 0.4% of samples. Only 0.9% of samples contained greater than 139 U. beta G activity in GCF was not related to gender or age. For the periodontitis patients, elevated enzyme activity (> or = 140 U) was most often associated with molar teeth, followed by maxillary bicuspids. Maxillary central incisors, and mandibular central and lateral incisors displayed the lowest frequency of elevated enzyme activity. The relationship of beta G activity to the traditional parameters of probing depth and bleeding on probing was assessed. For shallow sites (1.0-1.5 mm, 2.0-2.5 mm probing depth), the large majority of GCF samples contained low enzyme activity (90% of samples < 50 U). Descriptive indicators demonstrated a trend of increased beta G activity with increased probing depth. The median beta G activity shifted from 15 U for the shallowest sites (1.0-1.5 mm) to 127 U for the deepest sites (5-8 mm). However, this was due to a broadening of the distribution rather than representing a shift in the distribution profile.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential effects of adrenocorticotropic hormone on steroid hydroxylase activities in the inner and outer zones of the guinea pig adrenal cortex.

We have studied the effects of ACTH treatment on steroid hydroxylase activities in the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Animals received 5 or 10 U of ACTH daily for 6 days and enzyme activities were then assessed in isolated microsomal or mitochondrial preparations. In control animals, microsomal cytochrome P-450 concentrations were greater in the inner than outer zone, but mitochondrial P-450 levels were similar in the two zones. Microsomal 17 alpha-hydroxylase and mitochondrial 11 beta-hydroxylase activities were greater in the outer than inner zone, but microsomal 21-hydroxylase activity was greater in the inner zone. ACTH treatment decreased cytochrome P-450 concentrations in inner but not outer zone microsomes; mitochondrial P-450 levels were unaffected in both zones. ACTH caused a dose-dependent increase in inner zone 17 alpha-hydroxylase activity and decrease in 21-hydroxylase activity without affecting the activity of either enzyme in outer zone microsomes. ACTH also decreased 11 beta-hydroxylase activity in outer but not inner zone mitochondrial preparations. The net effect of ACTH treatment was to diminish the differences in steroid metabolism between the two zones. The results indicate that the effects of ACTH on steroid hydroxylase activities are both zone- and enzyme-dependent, suggesting the existence of multiple and independent regulatory mechanisms.

Mechanisms responsible for the thermal sensitivity of adrenal microsomal monooxygenases.

Studies were done to determine the mechanism(s) responsible for the thermal lability of adrenal microsomal monooxygenases. Preincubation of guinea pig adrenal microsomal suspensions at 37 degrees C caused large time-dependent declines in benzo(a)pyrene (BP) hydroxylase and benzphetamine (BZ) demethylase activities. Similar preincubations with hepatic microsomes had little effect on enzyme activities. The decreases in adrenal enzyme activities were completely prevented by co-incubation of microsomes with cytosol, but were not diminished by reduced glutathione, ascorbic acid, or bovine serum albumin. Partial protection was afforded by EDTA, suggesting that lipid peroxidation might be involved, but malonaldehyde production was not demonstrable and MnCl2, a potent inhibitor of lipid peroxidation, did not affect the decline in enzyme activities. The decreases in the rates of BP and BZ metabolism were prevented by including NADPH or NADP+ in the preincubation medium. The preincubation conditions causing losses of adrenal enzyme activities did not affect cytochrome P-450 concentrations or substrate binding to cytochromes P-450, as indicated by type I difference spectra. NADH-cytochrome c reductase activity also was not affected, but there were decreases in NADPH-cytochrome c reductase activity that were proportionately similar to the declines in drug-metabolizing activities. Direct assessment of NADPH-cytochrome P-450 reductase revealed similarly large decreases in enzyme activity resulting from preincubation of adrenal microsomes. The results demonstrate a need for extra caution when doing preincubation experiments with adrenal microsomal preparations, and suggest that the thermal lability of adrenal monooxygenases is attributable to effects at the active site of NADPH-cytochrome P-450 reductase.

Fluorometric assay for ADP-ribosylarginine cleavage enzymes.

A continuous fluorometric assay for enzyme activities which remove ADP-ribose linked to proteins at arginine was developed. The substrate analog, N alpha-dansyl-N omega-(1,N6-etheno-ADP-ribosyl)arginine methyl ester, was used to assay the catalytic activities of dinitrogenase reductase activating glycohydrolase from Rhodospirillum rubrum and nucleotide pyrophosphatase from Crotalus adamanteus. The assay is based on the increase in fluorescent emission by ethenoadenine accompanying the enzyme-catalyzed hydrolysis of the substrate. The assay has been used to detect activities of 10 fmol substrate cleaved per minute. The substrate anomerizes to give a 40:60 equilibrium of alpha:beta ribosylguanidinium anomers, allowing the determination of enzyme stereospecificity. The substrate was used to determine the kinetic parameters and products of the N-glycohydrolase and the pyrophosphatase.

Effects of cadmium in vitro on microsomal steroid metabolism in the inner and outer zones of the guinea pig adrenal cortex.

Studies were carried out to evaluate the effects of cadmium in vitro on microsomal steroid metabolism in the inner (zona reticularis) and outer (zona fasciculata and zona glomerulosa) zones of the guinea pig adrenal cortex. Microsomes from the inner zone have greater 21-hydroxylase than 17 alpha-hydroxylase activity, resulting in the conversion of progesterone primarily to 11-deoxycorticosterone and of 17 alpha-hydroxyprogesterone principally to its 21-hydroxylated metabolite, 11-deoxycortisol. Microsomes from the outer zones, by contrast, have far greater 17 alpha-hydroxylase and C17,20-lyase activities than 21-hydroxylase activity. As a result, progesterone is converted primarily to its 17-hydroxylated metabolite, 17 alpha-hydroxyprogesterone; and 17 alpha-hydroxyprogesterone is converted principally to delta 4-androstenedione, with only small amounts of 21-hydroxylated metabolites being produced. Addition of cadmium to incubations with inner zone microsomes causes concentration-dependent decreases in 21-hydroxylation and increases in 17 alpha-hydroxylase and C17,20-lyase activities, resulting in a pattern of steroid metabolism similar to that in normal outer zone microsomes. Cadmium similarly decreases 21-hydroxylation by outer zone microsomes but has no effect on the formation of 17-hydroxylated metabolites or on androgen (delta 4-androstenedione) production. In neither inner nor outer zone microsomes did cadmium affect cytochrome P-450 concentrations, steroid interactions with cytochrome(s) P-450, or NADPH-cytochrome P-450 reductase activities. The results indicate that cadmium produces both quantitative and qualitative changes in adrenal microsomal steroid metabolism and that the nature of the changes differs in the inner and outer adrenocortical zones. In inner zone microsomes, there appears to be a reciprocal relationship between 21-hydroxylase and 17 alpha-hydroxylase/C17,20-lyase activities which may influence the physiological function(s) of that zone.

N-glycohydrolysis of adenosine diphosphoribosyl arginine linkages by dinitrogenase reductase activating glycohydrolase (activating enzyme) from Rhodospirillum rubrum.

The reaction catalyzed by the activating enzyme for dinitrogenase reductase from Rhodospirillum rubrum has been studied using an ADP-ribosyl hexapeptide, obtained from proteolysis of inactive dinitrogenase reductase, and synthetic analogs such as N alpha-dansyl-N omega-ADP-ribosylarginine methyl ester. The activating enzyme catalyzed N-glycohydrolysis of the ribosyl-guanidinium linkage releasing ADP-ribose and regenerating an unmodified arginyl guanidinium group. Optimal glycohydrolysis of the low molecular weight substrates occurred at pH 6.6 and required 1 mM MnCl2, but did not require ATP. The ADP-ribosyl hexapeptide (Km 11 microM), N alpha-dansyl-N omega-ADP-ribosylarginine methyl ester (Km 12 microM), N alpha-dansyl-N omega-ADP-ribosylarginine (Km 12 microM), N alpha-dansyl-N omega-1,N6-etheno-ADP-ribosylarginine methyl ester (Km 11 microM), and N alpha-dansyl-N omega-GDP-ribosylarginine methyl ester (Km 11 microM) were comparable substrates. N omega-ADP-ribosylarginine (Km 2 mM) was a poor substrate, and the activating enzyme did not catalyze N-glycohydrolysis of N alpha-dansyl-N omega-5'-phosphoribosylarginine methyl ester or N alpha-dansyl-N omega-ribosylarginine methyl ester. 13C NMR of N alpha-tosyl-N omega-ADP-ribosylarginine methyl ester established that the activating enzyme specifically hydrolyzed the alpha-ribosyl-guanidinium linkage. The beta-linked anomer was hydrolyzed only after anomerization to the alpha configuration. We recommend [arginine(N omega-ADP-alpha-ribose)]dinitrogenase reductase N-glycohydrolase (dinitrogenase reductase activating) and dinitrogenase reductase activating glycohydrolase as the systematic and working names for the activating enzyme.

Studies on the activating enzyme for iron protein of nitrogenase from Rhodospirillum rubrum.

Removal of ADP-ribose from the iron protein of nitrogenase by activating enzyme resulted in the activation of the inactive iron protein. A radioassay that directly measured the initial velocity of the activation was developed using iron protein radiolabeled with either [8-3H]- or [G-32P]ADP-ribose. The release of radiolabeled ADP-ribose by activating enzyme was linearly correlated with the increase in the specific activity of the iron protein as measured by acetylene reduction. Both ATP and MnCl2 were required for the activation of inactive iron protein. The optimal ratio of [MnCl2]/[ATP] in the radioassay was 2:1, and the optimal concentrations were 4 mM and 2 mM for [MnCl2] and [ATP], respectively. The Km for inactive iron protein was 74 microM and the Vmax was 628 pmol of [32P] ADP-ribose released min-1 microgram of activating enzyme-1. Adenosine, cytidine, guanosine, or uridine mono-, di-, or triphosphates did not substitute for ATP in the activation of native iron protein. Activating enzyme removed ADP-ribose from oxygen-denatured iron protein in the absence of ATP. ADP, ADP-ribose, pyrophosphate, and high concentrations of NaCl inhibited activating enzyme activity.

Covalent modification of the iron protein of nitrogenase from Rhodospirillum rubrum by adenosine diphosphoribosylation of a specific arginine residue.

Nitrogenase in Rhodospirillum rubrum is inactivated in vivo by the covalent modification of the Fe protein with a nucleotide. The preparation of two modified peptides derived from proteolytic digestion of the inactive Fe protein is described. The modifying group is shown to be adenosine diphosphoribose, linked through the terminal ribose to a guanidino nitrogen of arginine. The structural features were established by using proton and phosphorus NMR, positive- and negative-ion fast atom bombardment mass spectrometry, and fast atom bombardment/collisionally activated decomposition mass spectrometry. Spectral methods along with chromatographic analysis and sequential degradation established the sequence of the modification site of Fe protein as Gly-Arg(ADR-ribose)-Gly-Val-Ile-Thr. This corresponds to the sequence in the Fe protein from Azotobacter vinelandii for amino acid residues 99 to 104.

Purification and properties of the heat-released nucleotide-modifying group from the inactive iron protein of nitrogenase from Rhodospirillum rubrum.

Nitrogenase in Rhodospirillum rubrum is regulated in vivo by the covalent modification of the Fe protein. This paper reports the isolation, purification, and properties of the modifying group that has been heat released from the Fe protein. The molecule is isolated from the heated mixture by binding to a boronate affinity column. Purification is achieved on an ion-exchange high-performance liquid chromatography column. Structural properties of the molecule have been investigated by using proton and phosphorus NMR, mass spectrometry, enzyme susceptibility, and chromatographic methods. The heat-released modifying group exhibits an unusual signal in the proton NMR spectrum at 1.26 ppm. The molecule also contains a functional group which can be reduced by borohydride. This group is lost on breakdown of the molecule or upon treatment of the molecule with 5'-nucleotidase. The identity of the base and the pentose of modifying group as adenine and ribose, respectively, is confirmed. Ratios of the known components of the modifying group are established.

Sites of action of cadmium in vitro on hepatic, adrenal, and pulmonary microsomal monooxygenases in guinea pigs.

Preincubation of hepatic, adrenal, or pulmonary microsomal preparations with cadmium produced time-dependent decreases in monooxygenase (benzphetamine demethylase, benzo(a)pyrene hydroxylase) activities. Addition of cadmium after the preincubation period had little or no effect on microsomal metabolism. As a result of preincubation with cadmium, hepatic cytochrome P-450 levels declined and the magnitude of the benzphetamine-induced type I spectral change in hepatic microsomes decreased. Cadmium also decreased hepatic NADPH-cytochrome c and NADPH-cytochrome P-450 reductase activities but had no effect on NADH-cytochrome c reductase activity. Cadmium similarly decreased cytochrome P-450 concentrations and NADPH-cytochrome c reductase activity in lung microsomes without affecting NADH-cytochrome c reductase activity. Preincubation of adrenal microsomes with cadmium had no effects on cytochrome P-450 levels, on the benzphetamine-induced type I spectrum, or on NADH-cytochrome c reductase activity. However, decreases in adrenal NADPH-cytochrome c and NADPH-cytochrome P-450 reductase activities resulted which closely paralleled the decline in adrenal monooxygenase activities. EDTA extraction of hepatic, adrenal, or pulmonary microsomes after the preincubation exposure removed about 95% of the cadmium but did not diminish the effects of the metal on microsomal monooxygenases. The results indicate that cadmium has somewhat varying sites of action on hepatic, adrenal, and pulmonary monooxygenases, but in all three tissues electron transfer to cytochrome P-450 is compromised. In addition, the effects of cadmium on microsomal metabolism persist fully even after removal of approximately 95% of the metal.

The duration of exposure of microsomal preparations to cadmium or zinc in vitro influences the inhibition of mono-oxygenases.

Preincubation of guinea pig hepatic, pulmonary, or adrenal microsomes with cadmium or zinc decreased mono-oxygenase [benzo(a)pyrene hydroxylase, benzphetamine demethylase] activities. Addition of the same concentrations of the metals to the microsomal suspensions after the preincubation period had little or no effect on enzyme activities. The decline in mono-oxygenase activities produced by cadmium or zinc was dependent on the length of the preincubation period as well as the concentration of metal present during the preincubation. In addition, the preincubation effects of both metals were temperature dependent; at temperatures between 4 and 37 degrees C, loss of enzyme activity increased with increasing temperature. Cadmium and zinc produced greater decreases in mono-oxygenase activities in pulmonary and adrenal microsomes than in hepatic microsomes. The results indicate that the duration of exposure of hepatic and extrahepatic microsomal preparations to cadmium or zinc in vitro is an important determinant of effects on mono-oxygenases.

Differential effects of adrenocorticotropic hormone on adrenal microsomal xenobiotic and steroid metabolism in guinea pigs.

The effects of adrenocorticotropic hormone (ACTH) administration to guinea pigs on the activities of adrenal microsomal monooxygenases were studied. ACTH treatment decreased the rates of adrenal benzphetamine (BZ) demethylation and benzo[a]pyrene (BP) hydroxylation but had no effect on the same reactions in hepatic microsomes. Adrenal microsomal steroid hydroxylation reactions were unaffected (21-hydroxylation) or stimulated (17 alpha-hydroxylation) by ACTH. Although ACTH treatment decreased adrenal BP hydroxylase activity, the relative quantity of the various BP metabolites, as determined by HPLC, did not change. Adrenal microsomal cytochrome P-450 concentrations were decreased by ACTH but proportionately less than the decreases in adrenal xenobiotic metabolism. The maximal type I spectral changes produced by xenobiotics in adrenal microsomes were decreased in size by ACTH treatment, but steroid-induced difference spectra were unaffected. The results indicate that ACTH selectively decreases the rates of adrenal xenobiotic metabolism, perhaps by producing a selective decline in the concentration(s) of those cytochromes P-450 involved in the metabolism of foreign compounds.

Metabolism of benzo [a] pyrene by guinea pig adrenal and hepatic microsomes.

Studies were carried out to compare the metabolism of benzo [a] pyrene (BP) by adrenal and hepatic microsomes obtained from adult male guinea pigs. Adrenal microsomes produced fluorescent metabolites (primarily phenols) approximately three to four times more rapidly than hepatic microsomes, but the differences in the rates were considerably smaller when total BP metabolism was assessed using an isotopic assay. The apparent discrepancy between the two assays is attributable to differences in the profiles of BP metabolites produced by adrenal and liver. Separation of metabolites by high pressure liquid chromatography revealed that adrenal microsomes converted BP to primarily a phenolic metabolite with a retention time identical to that of 3-hydroxy-BP. Liver microsomes, by contrast, produced approximately equal amounts of compounds co-chromatographing with 3-hydroxy-BP and BP-4,5-dihydrodiol. Small amounts of other metabolites were also produced by adrenal and hepatic microsomes. Liver microsomes catalyzed the conversion of BP to metabolites that became covalently bound to exogenous DNA. The amount of binding was dependent upon the duration of incubation and concentration of microsomal protein. Adrenal microsomes, by contrast, did not promote BP binding to DNA. Inhibition of microsomal epoxide hydratase activity with trichloropropene oxide (TCPO) blocked the formation of dihydrodiol metabolites of BP by adrenal and liver microsomes. In the presence of TCPO, liver microsomes produced large amounts of a BP metabolite co-chromatographing with BP-4,5-oxide. TCPO also increased the rate of production of DNA-binding metabolites by liver microsomes but had no effect on the formation of DNA-binding metabolites by adrenal microsomes. The results demonstrate major differences in the pathways of BP metabolism by guinea pig adrenal and hepatic microsomes. Although adrenal microsomes metabolize BP more rapidly than hepatic microsomes, far greater amounts of reactive metabolites are produced by the liver. Thus, adrenal metabolism of BP may be of little toxicological significance.

Comparative effects of cadmium, zinc, and lead in vitro on pulmonary, adrenal, and hepatic microsomal metabolism in the guinea pig.

The in vitro effects of Cd, Zn, and Pb on pulmonary, adrenal, and hepatic microsomal enzyme activities in guinea pigs were compared. Cd and Zn produced concentration-dependent (20-200 microM) decreases in benzphetamine demethylase and biphenyl hydroxylase activities in adrenal, liver, and lung. Pb had no significant effect on either enzyme in any of the tissues studied. Adrenal and pulmonary enzymes were more sensitive to the effects of Cd and Zn than were hepatic enzymes. Benzo[a]pyrene hydroxylase and ethoxycoumarin deethylase activities were decreased by Zn, Cd, and Pb in adrenal, liver, and lung microsomes. The inhibitory effect on benzo[a]pyrene and ethoxycoumarin metabolism were far greater than those on benzphetamine or biphenyl metabolism. The relative potencies of the metals as inhibitors of xenobiotic metabolism were Zn greater than Cd greater than Pb. Cd and Zn also inhibited steroid 21-hydroxylase activity in adrenal microsomes, but Pb had no effect on steroid metabolism. In addition, microsomal epoxide hydratase activity in adrenal, liver, and lung was inhibited by Cd but not by Zn or Pb. The results demonstrate that adrenal and pulmonary microsomal enzymes, like those in liver, are inhibited by various metals. Inhibition of mixed-function oxidases by metals in vitro is apparently not related to changes in cytochrome P-450 levels or substrate binding to cytochrome P-450. In addition, the actions of Cd, Zn, and Pb in each tissue are highly dependent on the substrates employed.