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The effect of inorganic lead on hepatic biochemical and ultrastructural changes produced by phenobarbital
101
Toxicoiogv Letters, 10 (1982) 101-108 Elsevier Biomedical Press
THE EFFECT OF INORGANIC LEAD ON HEPATIC BIOCHEMICAL ULTRASTRUCTURAL CHANGES PRODUCED BY PHENOBARBITAL
AND
JERRY J. HJELLE, ALPHONSE POKLIS* and VERNON W. FISCHER** School of Pharmacy, University of Colorado, Boulder, CO and St. Louis University School of Medicine, St. Louis, MO (U.S.A.) (Received July lOth, 1981) (Accepted July 13th, 1981)
SUMMARY Lead acetate (105 pmol/kg, i.p.) decreased rat hepatic cytochrome P-450 to 57% and 63% of control values when measured 24 and 48 h after lead administration, respectively. A large increase in urinary delta-aminolevulinic acid (U-ALA) was observed after lead treatment, indicating a depression of heme synthesis. In addition, lead treatment produced dilated cisternae of the smooth endoplasmic reticulum (SER). Phenobarbital (100 mg/kg, i.p.) produced an induction of cytochrome P-450, proliferation of the SER, and did not alter U-ALA content. Simultaneous lead and phenobarbital treatment produced a delayed but robust induction of cytochrome P-450, only a moderate rise in U-ALA, and a reduced proliferation of the SER of hepatocytes. Therefore, phenobarbital, an inducer of heme synthetic enzymes, is apparently capable of reversing lead-induced inhibition of heme synthesis as measured by hepatic cytochrome P-450 induction and U-ALA content.
INTRODUCTION
Lead produces a variety of biological and toxicological effects, most notably disruption of porphyrin synthesis. This effect is associated with decreases in hemecontaining constituents including the hepatic microsomal hemoprotein cytochrome *Author to whom reprint requests and all correspondence should be addressed: Departments of Pathology and Pharmacology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 (U.S.A.). **Departments of Anatomy and Pathology, St. Louis University School of Medicine, St. Louis, MO 63104 (U.S.A.). Abbreviations: RER, rough endoplasmic urinary delta-aminolevulinic acid. 0378-4274/82/0000-0000/$02.75
reticulum; SER, smooth endoplasmic
0 Elsevier Biomedical Press
reticulum; U-ALA,
102
P-450. Inorganic lead ions inhibit heme synthesis by decreasing the activity of the sensitive heme-synthetic enzymes [l , 21 delta-aminolevulinic acid dehydratase and ferrochelatase. In addition, the catabolism of heme is enhanced due to an increase in the de novo synthesis of hepatic heme oxygenase [3,4]. Pretreatment with lead ions has been shown to decrease the in vivo metabolism of cytochrome P-450 substrates zoxazolamine, hexobarbital and methohexital [5-81. Other studies have shown that in vivo lead pretreatment inhibits microsomal electron transport components (NADPH cytochrome c and NADPH cytochrome P-450 reductases) as well as decreases microsomal cytochrome P-450 and associated enzymatic activities [5-l 11. Significant decreases in hepatic cytochrome P-450 have been observed after acute doses as low as 10.5 pmol/g lead, i.p. [8]. Lead ions at high doses also produce ultrastructural changes in hepatocytes with a decrease in RER, dilation of SER and swelling of mitochondria [ 121. Phenobarbital treatment produces profound inductive effects on hepatic heme synthesis, microsomal protein and cytochrome P-450 content. Further, this barbiturate stimulates the proliferation of hepatic parenchymal SER [13]. Thus it was of interest to determine if lead, an inhibitor of heme synthesis, would decrease or retard the stimulatory effects produced by phenobarbital on hepatic cytochrome P-450 concentration and SER profileration. U-ALA concentrations, a measure of the biological effects of lead ions on heme synthesis, were measured to determine if phenobarbital possessed a protective effect against lead-induced depression in heme synthesis.
MATERIALS AND METHODS
Male Sprague-Dawley rats (Cox strain) weighing 85-130 g were supplied by Laboratory Supply Company, Indianapolis, IN. The rats were housed in pairs in stainless steel metabolic cages, exposed to a 12 h light-12 h dark cycle and were provided Purina@ Rat Chow and deionized water ad lib. Lead acetate (105 pmol/kg, i.p., Mallinckrodt) for injection was prepared in sterile normal saline by acidifying the solution with acetic acid to a pH of 5.1. Sodium phenobarbital (100 mg/kg, i.p. Merck) was dissolved in normal saline. Control animals received sterile normal saline (2 ml/kg, i.p.). Lead and phenobarbital, when administered simultaneously were injected in different sides of the peritoneum. All animals were fasted for 24 h before sacrifice. Rat liver microsomes were prepared using the method of Maze1 [14], except that removal of nuclei and mitochondria from a 25% homogenate was accomplished by centrifugation at 15000 x g for 15 min. The cytochrome P-450 and cytochrome Bs content of washed microsomes was determined using the methods of Omura and Sato [15] and Strittmatter and Velick
103
[ 161. Cytochrome P-450 concentrations were calculated by using simultaneous equations by the method of Imai and Sato [17]. The protein content of the microsomal suspensions was determined using the method of Lowry et al. [ 181 as modified by Miller [19] using Bovine Serum Albumin (Sigma Chemical Co.) as a standard. Pooled 24 h urine samples from two rats in the same experimental group were collected and stored in the dark at 4°C. The U-ALA content was determined as described by Lauwreys [20]. The method of standard additions was employed to correct for interfering substances present in each urine sample. For electron microscopy, small cubes of liver, 1 mm in extent, were removed from the left lateral lobe and were immediately fixed in ice-cold 3% glutaraldehyde in Sorensen’s buffer and postfixed in 2% osmium tetroxide. The tissues were then dehydrated in a graded ethanol series and block-stained with saturated uranyl acetate in 70% ethanol. Epon-embedded blocks were sectioned using a Sorvall Porter MTZ-B ultramicrotome; ultra-thin sections were viewed with an AEI Corinth 275 transmission electron microscope. Statistical evaluation (Student’s ttest) of the data was performed as described by Snedecor and Cochran [21] with a P
RESULTS
The effects of lead acetate on hepatic cytochromes P-450 and BS are presented in Fig. 1. No single treatment or combination of treatments had an effect on body weight. Lead acetate (105 pmol/kg, i.p.) resulted in significant decreases in hepatic cytochrome P-450 levels 24 and 48 h after treatment. Cytochrome BS content was markedly decreased 12 and 24 h after treatment. In addition, lead ions elicited an increase in the liver to body weight ratio (12 h) and microsomal protein (24 h). Lead pretreatment produced moderate dilation of the cisternae of the RER and SER of hepatocytes. In contrast, phenobarbital (100 mg/kg, i.p.) treatment produced the expected [ 131 induction of the cytochrome P-450 concentrations/mg microsomal protein without affecting cytochrome BS content. Increases were observed in liver to body weight ratios after 12, 24, and 48 h and in microsomal protein after 48 h. The simultaneous administration of lead with phenobarbital produced a decrease in hepatic cytochrome P-450 content at 12 h followed by a large increase in this hemoprotein at 24 and 48 h. Cytochrome BS content was not affected by the combined administration. This treatment did increase liver to body ratios (12, 24 and 48 h) and microsomal protein content (48 h). Thus, lead ions at a dose of 105 pmol/kg delayed but did not suppress the induction of cytochrome P-450 produced by a 100 mg/kg dose of phenobarbital. Table I shows the effect of lead ions or simultaneous injection of lead ions and phenobarbital on U-ALA concentrations. Measurement of U-ALA is widely
104
CytoChromQ P-450 200..
s I L--. Cont. 12
: Cont. 12 24 24 40 Time After Treatmcrt (hr)
48
Fig. 1. Effect of various treatments on hepatic cytochromes P-450 and &/mg rnicrosomal protein at different time intervals after agent administration. Each point represents the mean it SE. of 2-6 animals. Control values obtained from eleven observations were 1.30~ 0.02 nM cytochrome P-45O/mg microsomal protein and 0.80+0.03 nM cytochrome &/mg microsomal protein.
employed as a clinical test of lead exposure. Lead ions produced a significant increase in U-ALA over the first 24 h after treatment, with a larger increase observed in 24-48 h urine samples. Ph~nobarbi~l did not affect U-ALA levels. Phenobarbital treatment in combination with lead ion administration decreased the rise in U-ALA levels 24-48 h after simultaneous treatment. Therefore, U-ALA concentration in accordance with hepatic hemoprotein concentrations indicate that phenobarbital lessens the block in heme synthesis produced by lead ions 24 and 48 h after simultaneous treatment. Fig. 2 is an electron micrograph of a rat liver obtained 48 h after phenobarbital TABLE I EFFECT OF VARIOUS CONTENT
TREATME~S
ON URINARY
DELTA-AMINOLEVULINIC
Treatment Groupa
U-ALA &g/24 h urine/kg)
Saline Phenobarbital Lead acetate 24 h 48 h Phenobarbital 24h 48 h
29+4 23+4 57*5b 97 + 17” and lead acetate 47r4b 521t6bsc
aEach value represents the mean S.E. of 3-6 observations. bsignific~tly different (P
group at 48 h.
ACID
105
106
treatment, showing hepatocytes with marked dilation of the cisternae, increases in the content of the SER and small amounts of the RER. Simultaneous injection with lead ion produced an apparent retardation of the ultrastructural changes elicited by phenobarbital, since proliferation of the SER had not occurred to the same extent as in phenobarbital-treated hepatocytes (Fig. 3). Further, the RER is more prominent in the livers of animals receiving the combined treatment. These findings resemble ultrastructural changes produced earlier in the time course of the inductive process produced by phenobarbital. The microsomal protein concentrations per gram liver were not different between the phenobarbital or combined treatment groups 48 h after administration. Combined lead ion and phenobarbital treatment produced an initial decline followed by an induction of hepatic cytochrome P-450 concentrations. Phenobarbital apparently reversed the inhibition of heme synthesis by lead ions thereby decreasing the rise in U-ALA content.
Fig. 3. Liver, 48 h after simultaneous lead and phenobarbital injection; slight proliferation of smooth (double arrows) or rough endoplasmic reticulum {arrows); electron micrograph, x 10000.
107
DISCUSSION
Lead ions decreased cytochrome P-450 concentrations with a time course consistent with the reported half-life of 22 h for rat liver cytochrome P-450 [lo]. In contrast to the findings of Chow and Cornish [IO] we did observe a decline in microsomal cytochrome BS content after lead ion administration. Phenobarbital treatment in combination with lead ions did not alter cytochrome BS concentrations. Simultaneous administration of lead ions and phenobarbital produced an initial decrease in microsomal cytochrome P-450 content at 12 h followed by a strong induction 24 and 48 h after treatment. These findings agree well with those of Maxwell [9] and Chow and Cornish [IO]. We have shown that associated with an induction of microsomal cytochrome P-450 was a lessening of lead-induced whole animal heme synthesis blockade, as measured by a decrease in U-ALA concentration. Inhibition of heme synthesis at the delta-aminolevulinic acid dehydratase step is very sensitive to free lead ion concentration. Granick et al. [l] reported that the log of delta-aminolevulinic acid dehydratase activity in red blood cells correlates inversely with blood lead concentration. Phenobarbital treatment may have altered the whole animal distribution and excretion of lead as well as decreased free lead ion concentrations in the liver. The rate-limiting enzyme of the heme synthetic pathway is delta-aminolevulinic acid synthetase, an enzyme which is induced by phenobarbital treatment and by low free heme concentration (i.e., an effect produced by inorganic lead ions). Maxwell [9] reported that synthetase activities responded synergistically following combined treatment when compared to lead ion or phenobarbital treatment alone. Thus, relatively small decreases in free lead on concentration may have profound effects on the degree of inhibition of heme synthesis at the dehydratase step and allow for normal or increased heme synthesis due to the large induction of synthetase activity. Ultrastructurally, lead ion produced the previously observed dilation of the cisternae of the SER of hepatocytes [ 121. Combined treatment produced a moderate increase in the content of SER of hepatocytes with RER components remaining in quantity 48 h after treatment. Microsomes prepared from these livers possessed high concentrations of cytochrome P-450. It appears that combined treatment decreased the large proliferation of the SER of the hepatocyte induced by phenobarbital treatment alone and appeared to preserve the protein synthetic machinery. In summary, lead ions could not block cytochrome P-450 induction 48 h after simultaneous administration with phenobarbital, but did decrease the extent of SER proliferation.
108
REFERENCES 1 J.L. Granick, S. Sassa, S. Granick, R.D. Levere and A. Kappas, Studies on lead poisoning, II. Correlation between the ratio of activated to inactivated delta-aminolevulinic acid dehydratase of whole blood and the blood lead level, Biochem. Med., 8 (1973) 149. 2 G.S. Wagner and T.R. Tephly, The possible role of copper in the regulation of heme biosynthesis through ferrochelatase, Adv. Exp. Med. Biol., 58 (1975) 343. 3 M.D. Maines and A. Kappas, The induction of heme oxidation in various tissues by trace metals: evidence for the catabolism of endogenous heme by hepatic oxygenase, Ann. Clin. Res., 8 (1976) 39. 4 M.D. Maines and A. Kappas, Metals as regulators of heme metabolism, Science, 198 (1977) 1215. 5 A.P. Scoppa, M. Roumengous and W. Penning, Hepatic drug metabolism activity in lead poisoned rats, Experientia, 29 (1971) 15. 6 A.P. Alvares, S. Leigh, J. Cohn and A. Kappas, Lead and methyl mercury: effects of acute exposure on cytochrome P-450 and the mixed function oxidase system in the liver, J. Exp. Med., 155 (1972) 1406. 7 A.P. Alvares, A. Fishbein, S. Sassa, K.E. Anderson and A. Kappas, Lead intoxication: effects on cytochrome P-450 mediated hepatic oxidations, Clin. Pharmacol. Ther., 19 (1976) 183. 8 J. J. Hjelle, M. Kemal and A. Poklis, Effects of acute inorganic lead administration on in vivo drug metabolism and cytochrome P-450 in the rat, Toxicol. Lett., 2 (1978) 361. 9 J.D. Maxwell, Enzyme induction and drug sensitivity in hereditary hepatic porphyria, in A. Richens and F.P. Woodford (Eds.), Anticonvulsant Drugs and Enzyme Induction, Excerpta Medica, Amsterdam, 1976, pp. 137-145. 10 C.P. Chow and H.H. Cornish, Effects of lead on the induction of hepatic microsomal enzymes by phenobarbital and 3,4-benzpyrene, Toxicol. Appl. Pharmacol., 43 (1978) 219. 11 D.L. Eaton, N.H. Stacey, K.L. Wong and CD. Klaassen, Dose response effects of various metal ions on rat liver metallothionein glutathione, heme oxygenase and cytochrome P-450, Toxicol. Appl. Pharmacol., 55 (1980) 393. 12 E.O. Hoffmann, R.A. Trejo, N.R. DiLuzio and J. Lamberty, Ultrastructural alterations of liver and spleen following acute lead administration in rats, Exp. Mol. Pathol., 17 (1970) 159. 13 H.S. Marver, The role of heme in the synthesis and repression of microsomal protein, in J.R. Gillette, A.H. Cooney, G.J. Cosmides, R.W. Estabrook, J.R. Fouts and G.J. Mannering (Eds.), Microsomes and Drug Oxidation, Academic Press, New York, 1969, pp. 495-515. 14 P. Mazel, General principles and procedures for drug metabolism in vitro, in B.N. LaDu, H.G. Mandel and E.L. Way (Eds.), Fundamentals of Drug Metabolism and Drug Disposition, Williams and Wilkins, Baltimore, 1971, pp. 546-590. 15 T. Omura and R. Sato, The carbon monoxide-binding pigment of liver, I. Evidence for its hemoprotein nature, J. Biol. Chem., 239 (1964) 2370. 16 P. Strittmatter and S.F. Velick, The isolation and properties of microsomal cytochrome, J. Biol. Chem., 22 (1956) 253. 17 Y. Imai and R. Sato, Dual effect of ethyl isocyanide on drug hydroxylation by liver microsomes, J. Biochem., 63 (1968) 380. 18 O.H. Lowry, H.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with Folin-phenol reagent, J. Biol. Chem., 193 (1951) 265. 19 G.L. Miller, Protein determination for a large number of samples, Anal. Chem., 31 (1959) 964. 20 R. Lauwreys, R. Delbroek and M.D. Veims, Automated analysis of delta-aminolevulinic acid in urine, Clin. Chem. Acta, 40 (1972) 443. 21 G.W. Snedecor and W.G. Cochran, Statistical Methods, Iowa State University Press, Ames, IA, 1967, p. 59.
Toxicoiogv Letters, 10 (1982) 101-108 Elsevier Biomedical Press
THE EFFECT OF INORGANIC LEAD ON HEPATIC BIOCHEMICAL ULTRASTRUCTURAL CHANGES PRODUCED BY PHENOBARBITAL
AND
JERRY J. HJELLE, ALPHONSE POKLIS* and VERNON W. FISCHER** School of Pharmacy, University of Colorado, Boulder, CO and St. Louis University School of Medicine, St. Louis, MO (U.S.A.) (Received July lOth, 1981) (Accepted July 13th, 1981)
SUMMARY Lead acetate (105 pmol/kg, i.p.) decreased rat hepatic cytochrome P-450 to 57% and 63% of control values when measured 24 and 48 h after lead administration, respectively. A large increase in urinary delta-aminolevulinic acid (U-ALA) was observed after lead treatment, indicating a depression of heme synthesis. In addition, lead treatment produced dilated cisternae of the smooth endoplasmic reticulum (SER). Phenobarbital (100 mg/kg, i.p.) produced an induction of cytochrome P-450, proliferation of the SER, and did not alter U-ALA content. Simultaneous lead and phenobarbital treatment produced a delayed but robust induction of cytochrome P-450, only a moderate rise in U-ALA, and a reduced proliferation of the SER of hepatocytes. Therefore, phenobarbital, an inducer of heme synthetic enzymes, is apparently capable of reversing lead-induced inhibition of heme synthesis as measured by hepatic cytochrome P-450 induction and U-ALA content.
INTRODUCTION
Lead produces a variety of biological and toxicological effects, most notably disruption of porphyrin synthesis. This effect is associated with decreases in hemecontaining constituents including the hepatic microsomal hemoprotein cytochrome *Author to whom reprint requests and all correspondence should be addressed: Departments of Pathology and Pharmacology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 (U.S.A.). **Departments of Anatomy and Pathology, St. Louis University School of Medicine, St. Louis, MO 63104 (U.S.A.). Abbreviations: RER, rough endoplasmic urinary delta-aminolevulinic acid. 0378-4274/82/0000-0000/$02.75
reticulum; SER, smooth endoplasmic
0 Elsevier Biomedical Press
reticulum; U-ALA,
102
P-450. Inorganic lead ions inhibit heme synthesis by decreasing the activity of the sensitive heme-synthetic enzymes [l , 21 delta-aminolevulinic acid dehydratase and ferrochelatase. In addition, the catabolism of heme is enhanced due to an increase in the de novo synthesis of hepatic heme oxygenase [3,4]. Pretreatment with lead ions has been shown to decrease the in vivo metabolism of cytochrome P-450 substrates zoxazolamine, hexobarbital and methohexital [5-81. Other studies have shown that in vivo lead pretreatment inhibits microsomal electron transport components (NADPH cytochrome c and NADPH cytochrome P-450 reductases) as well as decreases microsomal cytochrome P-450 and associated enzymatic activities [5-l 11. Significant decreases in hepatic cytochrome P-450 have been observed after acute doses as low as 10.5 pmol/g lead, i.p. [8]. Lead ions at high doses also produce ultrastructural changes in hepatocytes with a decrease in RER, dilation of SER and swelling of mitochondria [ 121. Phenobarbital treatment produces profound inductive effects on hepatic heme synthesis, microsomal protein and cytochrome P-450 content. Further, this barbiturate stimulates the proliferation of hepatic parenchymal SER [13]. Thus it was of interest to determine if lead, an inhibitor of heme synthesis, would decrease or retard the stimulatory effects produced by phenobarbital on hepatic cytochrome P-450 concentration and SER profileration. U-ALA concentrations, a measure of the biological effects of lead ions on heme synthesis, were measured to determine if phenobarbital possessed a protective effect against lead-induced depression in heme synthesis.
MATERIALS AND METHODS
Male Sprague-Dawley rats (Cox strain) weighing 85-130 g were supplied by Laboratory Supply Company, Indianapolis, IN. The rats were housed in pairs in stainless steel metabolic cages, exposed to a 12 h light-12 h dark cycle and were provided Purina@ Rat Chow and deionized water ad lib. Lead acetate (105 pmol/kg, i.p., Mallinckrodt) for injection was prepared in sterile normal saline by acidifying the solution with acetic acid to a pH of 5.1. Sodium phenobarbital (100 mg/kg, i.p. Merck) was dissolved in normal saline. Control animals received sterile normal saline (2 ml/kg, i.p.). Lead and phenobarbital, when administered simultaneously were injected in different sides of the peritoneum. All animals were fasted for 24 h before sacrifice. Rat liver microsomes were prepared using the method of Maze1 [14], except that removal of nuclei and mitochondria from a 25% homogenate was accomplished by centrifugation at 15000 x g for 15 min. The cytochrome P-450 and cytochrome Bs content of washed microsomes was determined using the methods of Omura and Sato [15] and Strittmatter and Velick
103
[ 161. Cytochrome P-450 concentrations were calculated by using simultaneous equations by the method of Imai and Sato [17]. The protein content of the microsomal suspensions was determined using the method of Lowry et al. [ 181 as modified by Miller [19] using Bovine Serum Albumin (Sigma Chemical Co.) as a standard. Pooled 24 h urine samples from two rats in the same experimental group were collected and stored in the dark at 4°C. The U-ALA content was determined as described by Lauwreys [20]. The method of standard additions was employed to correct for interfering substances present in each urine sample. For electron microscopy, small cubes of liver, 1 mm in extent, were removed from the left lateral lobe and were immediately fixed in ice-cold 3% glutaraldehyde in Sorensen’s buffer and postfixed in 2% osmium tetroxide. The tissues were then dehydrated in a graded ethanol series and block-stained with saturated uranyl acetate in 70% ethanol. Epon-embedded blocks were sectioned using a Sorvall Porter MTZ-B ultramicrotome; ultra-thin sections were viewed with an AEI Corinth 275 transmission electron microscope. Statistical evaluation (Student’s ttest) of the data was performed as described by Snedecor and Cochran [21] with a P
RESULTS
The effects of lead acetate on hepatic cytochromes P-450 and BS are presented in Fig. 1. No single treatment or combination of treatments had an effect on body weight. Lead acetate (105 pmol/kg, i.p.) resulted in significant decreases in hepatic cytochrome P-450 levels 24 and 48 h after treatment. Cytochrome BS content was markedly decreased 12 and 24 h after treatment. In addition, lead ions elicited an increase in the liver to body weight ratio (12 h) and microsomal protein (24 h). Lead pretreatment produced moderate dilation of the cisternae of the RER and SER of hepatocytes. In contrast, phenobarbital (100 mg/kg, i.p.) treatment produced the expected [ 131 induction of the cytochrome P-450 concentrations/mg microsomal protein without affecting cytochrome BS content. Increases were observed in liver to body weight ratios after 12, 24, and 48 h and in microsomal protein after 48 h. The simultaneous administration of lead with phenobarbital produced a decrease in hepatic cytochrome P-450 content at 12 h followed by a large increase in this hemoprotein at 24 and 48 h. Cytochrome BS content was not affected by the combined administration. This treatment did increase liver to body ratios (12, 24 and 48 h) and microsomal protein content (48 h). Thus, lead ions at a dose of 105 pmol/kg delayed but did not suppress the induction of cytochrome P-450 produced by a 100 mg/kg dose of phenobarbital. Table I shows the effect of lead ions or simultaneous injection of lead ions and phenobarbital on U-ALA concentrations. Measurement of U-ALA is widely
104
CytoChromQ P-450 200..
s I L--. Cont. 12
: Cont. 12 24 24 40 Time After Treatmcrt (hr)
48
Fig. 1. Effect of various treatments on hepatic cytochromes P-450 and &/mg rnicrosomal protein at different time intervals after agent administration. Each point represents the mean it SE. of 2-6 animals. Control values obtained from eleven observations were 1.30~ 0.02 nM cytochrome P-45O/mg microsomal protein and 0.80+0.03 nM cytochrome &/mg microsomal protein.
employed as a clinical test of lead exposure. Lead ions produced a significant increase in U-ALA over the first 24 h after treatment, with a larger increase observed in 24-48 h urine samples. Ph~nobarbi~l did not affect U-ALA levels. Phenobarbital treatment in combination with lead ion administration decreased the rise in U-ALA levels 24-48 h after simultaneous treatment. Therefore, U-ALA concentration in accordance with hepatic hemoprotein concentrations indicate that phenobarbital lessens the block in heme synthesis produced by lead ions 24 and 48 h after simultaneous treatment. Fig. 2 is an electron micrograph of a rat liver obtained 48 h after phenobarbital TABLE I EFFECT OF VARIOUS CONTENT
TREATME~S
ON URINARY
DELTA-AMINOLEVULINIC
Treatment Groupa
U-ALA &g/24 h urine/kg)
Saline Phenobarbital Lead acetate 24 h 48 h Phenobarbital 24h 48 h
29+4 23+4 57*5b 97 + 17” and lead acetate 47r4b 521t6bsc
aEach value represents the mean S.E. of 3-6 observations. bsignific~tly different (P
group at 48 h.
ACID
105
106
treatment, showing hepatocytes with marked dilation of the cisternae, increases in the content of the SER and small amounts of the RER. Simultaneous injection with lead ion produced an apparent retardation of the ultrastructural changes elicited by phenobarbital, since proliferation of the SER had not occurred to the same extent as in phenobarbital-treated hepatocytes (Fig. 3). Further, the RER is more prominent in the livers of animals receiving the combined treatment. These findings resemble ultrastructural changes produced earlier in the time course of the inductive process produced by phenobarbital. The microsomal protein concentrations per gram liver were not different between the phenobarbital or combined treatment groups 48 h after administration. Combined lead ion and phenobarbital treatment produced an initial decline followed by an induction of hepatic cytochrome P-450 concentrations. Phenobarbital apparently reversed the inhibition of heme synthesis by lead ions thereby decreasing the rise in U-ALA content.
Fig. 3. Liver, 48 h after simultaneous lead and phenobarbital injection; slight proliferation of smooth (double arrows) or rough endoplasmic reticulum {arrows); electron micrograph, x 10000.
107
DISCUSSION
Lead ions decreased cytochrome P-450 concentrations with a time course consistent with the reported half-life of 22 h for rat liver cytochrome P-450 [lo]. In contrast to the findings of Chow and Cornish [IO] we did observe a decline in microsomal cytochrome BS content after lead ion administration. Phenobarbital treatment in combination with lead ions did not alter cytochrome BS concentrations. Simultaneous administration of lead ions and phenobarbital produced an initial decrease in microsomal cytochrome P-450 content at 12 h followed by a strong induction 24 and 48 h after treatment. These findings agree well with those of Maxwell [9] and Chow and Cornish [IO]. We have shown that associated with an induction of microsomal cytochrome P-450 was a lessening of lead-induced whole animal heme synthesis blockade, as measured by a decrease in U-ALA concentration. Inhibition of heme synthesis at the delta-aminolevulinic acid dehydratase step is very sensitive to free lead ion concentration. Granick et al. [l] reported that the log of delta-aminolevulinic acid dehydratase activity in red blood cells correlates inversely with blood lead concentration. Phenobarbital treatment may have altered the whole animal distribution and excretion of lead as well as decreased free lead ion concentrations in the liver. The rate-limiting enzyme of the heme synthetic pathway is delta-aminolevulinic acid synthetase, an enzyme which is induced by phenobarbital treatment and by low free heme concentration (i.e., an effect produced by inorganic lead ions). Maxwell [9] reported that synthetase activities responded synergistically following combined treatment when compared to lead ion or phenobarbital treatment alone. Thus, relatively small decreases in free lead on concentration may have profound effects on the degree of inhibition of heme synthesis at the dehydratase step and allow for normal or increased heme synthesis due to the large induction of synthetase activity. Ultrastructurally, lead ion produced the previously observed dilation of the cisternae of the SER of hepatocytes [ 121. Combined treatment produced a moderate increase in the content of SER of hepatocytes with RER components remaining in quantity 48 h after treatment. Microsomes prepared from these livers possessed high concentrations of cytochrome P-450. It appears that combined treatment decreased the large proliferation of the SER of the hepatocyte induced by phenobarbital treatment alone and appeared to preserve the protein synthetic machinery. In summary, lead ions could not block cytochrome P-450 induction 48 h after simultaneous administration with phenobarbital, but did decrease the extent of SER proliferation.
108
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