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The integrity of liver protein synthesis in male rats treated with 1,2-dibromo-3-chloropropane
Toxicology Letters, 12 (1982) 101-108 Elsevier Biomedical Press
101
THE INTEGRITY OF LIVER PROTEIN SYNTHESIS IN MALE RATS TREATED WITH 1,2-DIBROMO-3-CHLOROPROPANE
D.E. MOODY, GA. CLAWSON and E.A. SMUCKLER Department
of Pathology,
University of California,
School of Medicine,
San Francisco,
CA 94143
(U.S.A.)
(Received January 7th, 1982) (Accepted March 2nd, 1982)
SUMMARY Male rats treated with a single dose of 1,2-dibromo-3-chloropropane (DBCP) were tested for their ability to carry out the synthesis of liver proteins. In animals treated for 12 h, we found no changes in the uptake of [14C]orotic acid into liver RNA or the uptake of [3H]leucine into liver or serum protein. Uptake of [jH]leucine into the soluble fraction of the enlarged liver increased in proportion to liver size, while the uptake of [14C]orotic acid was unchanged. Examination of the ultrastructure of liver cells from rats treated for 12, 24, or 48 h revealed that the structure of the rough and smooth endoplasmic reticulum (RER; SER) were modified. An absence of ordered stacks of the RER and the presence of tangled nets of SER were noted.
INTRODUCTION
In test animals, acute exposure to DBCP has been found to result in damage to testes, kidney, lung, and liver [l, 21. Chronic exposure produces tumors in forestomach, mammary gland, lung, and nasopharynx [3-51, and effects the reproductive capacity of male animals [6]. In addition, DBCP produced mutagenesis in bacterial systems [7-g]. The reproductive lesion has also been found in men occupationally exposed to this compound [lo, 111. The mechanism of the reproductive failure is as yet unknown; an alteration in steroid metabolism is a plausible explanation [ 121. The cytochrome P-UO-dependent microsomal mixed-function oxidases have been
Abbreviations: DBCP, 1,2-dibromo-3-chloropropane; ER, endoplasmic reticulum; RER, endoplasmic reticulum; SER, smooth endoplasmic reticulum; TCA, trichloroacetic acid. 0378-4274/82/0000-0000/$02.75
0 Elsevier Biomedical Press
rough
102
found to play a role in many facets of steroid metabolism including their synthesis and catabolism [13]. Whether alterations in the activity of this system are reflected in alterations of steroid function has not been thoroughly examined. We have recently found that a single treatment with DBCP leads to a decrease in hepatic cytochrome P-450 [14] and have presented preliminary evidence that this decrease occurs in microsomes from testis, lung, kidney, and intestinal mucosa [15]. The decrease in testicular microsomal cytochrome P-450 has also been reported by Tofilon and coworkers [16]. These authors also found an alteration in the synthesis of heme in testicular tissue. One manner in which toxins can cause a decrease in protein levels is through a reduction in protein synthesis, either at the level of transcription (RNA synthesis) or translation (protein synthesis). Several toxins are known to have gross effects on these cellular actions [17-191. In the current study, we have tested the effect of DBCP on several parameters of protein synthesis in liver cells. We found that DBCP had little or no effect on the overall process of protein synthesis. These findings, and previous findings on the response of microsomal components to alkyl halides [20], suggest that the action of DBCP on microsomal cytochrome P-450 is specific and not the result of a major lesion in the hepatocyte. METHODS
Male Sprague-Dawley rats (caesarian delivered, Charles River, Wilmington, MA) were housed in pairs in steel screen-bottomed cages with food and water provided ad lib. Food was removed 18 h prior to killing. DBCP (technical grade, Occidental Chemical Co., Lathrop, CA) was dissolved in mineral oil (2% v/v) and 0.1 ml/kg administered by oral gavage. Control rats received an equal volume of mineral oil (5.0 ml/kg). 2 h prior to killing, either 250 &i/kg (4, 5)-[3H]leucine (New England Nuclear, 60 Ci/mmol) or 30 &i/kg (6)-[14C]orotic acid (New England Nuclear, 28 mCi/mmol) were injected via the tail vein. At the times specified, animals were anesthetized under light ether, blood was collected by cardiac puncture, the livers were perfused with 0.9% saline, removed into tared beakers, minced, weighed, and homogenized in 3.4 ~01s. of 0.1 M potassium phosphate (pH 7.4). An aliquot of the homogenate was precipitated with an equal volume of 20% TCA. The supernatant was collected for determination of the unincorporated label taken up by the liver. The pellet was then extracted as described by Sadowski and Steiner (211, which first removes lipids and then successively extracts RNA, DNA, and protein. Aliquots from the initial TCA supernate and the latter extracts were dissolved in protosol, suspended in aquasol, and the radioactivity was determined by liquid scintillation counting. The contents of RNA [22], DNA [23], and protein [24] in the respective extracts were determined by the methods cited. Sections were taken from livers after perfusion and fixed in 1% osmium tetroxide in S-collidine buffer (pH 7.4) prior to preparation for examination by electron
103
microscopy. Serum was prepared from the blood samples and separated into albumin and globulin fractions as previously described by extraction of albumin from TCA precipitates of serum with methanol [25]. RESULTS
The livers of male rats increase in weight following treatment with DBCP [14]. Relative liver weights were 1.28 times greater than control livers after 12 h of treatment (Table I). The content of total liver DNA did not change signi~cantly following treatment, but liver RNA and protein increased in a parallel manner to the liver size. These results suggest that the increase in liver size arises predominantly from cellular hypertrophy rather than cell division. The uptake of the radioactively labeled precursors into the liver was estimated from the radioactivity recovered in the supernates after precipitation of the liver TABLE I THE EFFECT OF 12-h TREATMENT WITH DBCP ONLY LIVER SIZE, DNA, AND RNA, AND LIVER AND SERUM PROTEIN CONTENT AND THE INCORPORATION OF LABELED PRECURSORS INTO EXTRACED RNA AND PROTEINa Sample Liver weight g/100 g body wt TCA supernate [t*C]CPM/lOO g body weight (lO-6) [3H]CPM/i~ g body weight DNA extract mg/lOO g body weight RNA extract mg/lOO g body weight [‘%]CPM/pg RNA Protein extract mg/lGil g body weight [sH]CPM/mg protein Serum albumin (mg/ml) [sH]CPM/mg protein Serum globulin (mg/ml) [sH]CPM/mg protein
Control
DBCP
3.55 +
0.24
4.56 +z 0.25b
1.19* 78.6 +
0.17 9.5
1.19 f 0.23~ 93.5 + 17.4”
6.56 +
0.8
10.5 f. 18.1 rf
1.9 6.6
368 310.8 2.19 979 7.20 816
f 78 f 33.3 zk 0.58 + 267 + 0.59 t 156
7.09 t
1.18c
12.1 + 0.4” 15.7 + 4.4 468 f 6b 322.3 zk 34.3 2.32 zk 0.39 984 f 80 7.70& 1.43 789 f 86
aExtracts of liver homogenates and serum were prepared from rats Iabeled with [3H]leucine ([sH]CPM) or [tK]orotic acid ([t4c]CPM), as described under METHODS. Values are the mean + SD for at least 4 animals. Due to the change in liver size after treatment with DBCP, the values were routinely expressed per g liver and per total liver of a 100-g rat. Only the latter values are presented, but significant differences are noted for both cases. bsignificantly different from controls when values are calculated for total liver/l00 g body weight, P-20.05. %SigniBcantly different from control when values calculated/g liver, P < 0.05.
104
homogenates with TCA. While the uptake of [i4C]orotic acid into the total liver was not significantly changed (Table I), the uptake of t3H]leucine increased along with the increase in liver size (Table I). The mechanism of entry of these two compounds may, therefore, respond differently to treatment with DBCP. Liver RNA and protein were extracted from TCA precipitates of liver homogenates and the specific radioactivity of the two fractions was determined. In animals prelabeled with [14C]orotic acid or [3H]leucine no significant change was found in their incorporation into extracted RNA or protein, respectively (Table I). Furthermore, the incorporation of label into serum-albumin fraction, a protein synthesized and secreted from the liver, and the serum-globulin fraction, proteins of a heterogeneous origin, were not affected. The synthesis of RNA and protein in livers of rats does not appear to be effected 12 h after treatment with DBCP. Sections of livers from rats treated for 12,24, and 48 h with DBCP were examined by light and electron microscopy. The livers of our control rats were not different from previous descriptions of Sprague-Dawley male rats. At 12, 24, or 48 h following DBCP the only consistent light microscopic finding was a dispersal of the cytoplasmic basophilia. Ultrastructural examination revealed that the ER was modified (Fig. 1). Stacked arrays of RER were almost absent. Ribosomes remained attached to the membrane and whorls and aggregates of cytosolic polysomes were detected. Dilation of the cisternae of both the RER and SER was also apparent, and areas of the SER were aggregated in tangled nets (Fig. 1). In addition, the hepatocytes from animals tested with DBCP contained a number of non membranebound lipid droplets. It was observed that the mitochondrial cristae did not always penetrate to the middle of the organelle when compared to controls. The plasmalemma, nuclear envelope, nucleoli, and other organelles, were not different from controls. DISCUSSION
Several reports on the acute and chronic effects of DBCP on male rats have mentioned alterations in liver cells which include liver enlargement, congestion, and focal centrilobular necrosis [l-5]. In previous studies we have noted that treatment with DBCP causes a decrease in hepatic microsomal cytochromes [14]. This is associated with a change in the relative content of microsomal fatty acids, particularly arachidonic and linoleic acids [20]. Several compounds which also cause a decrease in hepatic microsomal cytochrome P-450 cause a decrease in the synthesis of hepatic proteins [17-191. This latter defect may in part be responsible for the decline in cytochrome content. The results of the current study suggest that DBCP does not effect an overall decrease in protein synthesis either by a direct action on the incorporation of amino acids or indirectly, through an effect on the synthesis of RNA. These studies, however, cannot rule out the action of DBCP on the synthesis of specific messengers
Fig. 1. Electron micrographs of sections of liver tissue taken from (A) control male rat or (B) a male rat 12 h after treatment with 0.1 ml/kg DBCP. The photos demonstrate the difference in the: spaltial arrangement of the RER, which is more commonly dispersed in the treated animals, and the pre sencle of tangled nets of SER. Lipid droplets (Id) are present in hepatocytes from the treated rats. Notice that the other organelles are not markedly effected by the treatment. The bar represents 1 pm.
(i.e., the messenger for cytochrome P-450 apoprotein) or the regulation of its translation. Another point of regulation for the synthesis of microsomal cytochromes is the turnover of the heme moiety. Tofilon and coworkers [16] have described a decrease in the uptake of the heme precursor, b-aminolevulinic acid, into testicular microsomal heme. This was associated with a decrease in cytochrome P-450 and changes in certain heme synthesis enzymes. Preliminary results from our
107
laboratory show that a similar defect in heme synthesis may occur in liver tissue, as a 15% decrease in the uptake of d-aminolevulinic acid was found after 12 h of treatment 1261. At this point the importance of the decrease in cytochrome P-450 to the toxic effects of DBCP are not known. Due to the role of cytochrome P-450 in the metabolism of numerous endogenous lipids, including steroid hormones, the decrease may be linked to subsequent physiological changes which follow exposure to DBCP. As DBCP and other alkyl halides, which also effect cytochrome P-450 levels [20], are known to effect the reproductive capacity of test animals and humans [9-l 11, this may prove to be an important step in the toxic action of these compounds. ACKNOWLEDGEMENT
This work was supported by USPHS Grant AM-19843 and CA-21141, and by a Monsanto Fund Fellowship in Toxicology. REFERENCES 1 T.R. Torkelson, S.E. Sadek, V.K. Rowe, J.K. Kodama, H.H. Anderson, G.S. Loquvam and C.H. Hine, Toxicologic investigations of 1,2-dibromo-3-chloropropane, Toxicol. Appl. Pharmacol., 3 (1961) 545-559. 2 W.M. Kluwe, Acute toxicity of 1,2-dibromo-3-chloropropane in the F344 male rat, I. Dose-response relationships and differences in routes of exposure, Toxicol. Appl. Pharmacol., 59 (1981) 71-83. 3 W.A. Olson, R.T. Habermann, E.K. Weisburger, J.M. Ward and J.H. Weisburger. Induction of stomach cancer in rats and mice by halogenated aliphatic fumigants, J. Natl. Cancer Inst., 51 (1973) 1993-1995. 4 G. Reznik, S.F. Stinson and J.M. Ward, Lung tumors induced by chronic inhalation of 1,2-dibromo3-chloropropane in B&sFi mice, Cancer Lett., 10 (1980) 339-342. 5 G. Reznik, B. Ulland, S.F. Stinson and J.M. Ward, Morphology and sex-dependent manifestation of nasal tumors in B6C3Fl mice after chronic inhalation of 1,2-dibromo-3-chloropropane, J. Cancer Res. Clin. Oncol., 98 (1980) 75-83. 6 K.S. Rao, F.J. Murray, A.A. Crawford, J.A. John, W.J. Potts, B.A. Schwetz, J.D. Burek and C.M. Parker, Effects of inhaled, 1,2-dibromo-3-chloropropane (DBCP) on the semen of rabbits and the fertility of male and female rats, Toxicol. Appl. Pharmacol., 48 (1979) Al37 (Abst). 7 H.S. Rosenkranz, Genetic activity of 1,2-dibromo-3-chloropropane. A widely-used fumigant, Bull. Environ. Contam. Toxicol., 14 (1975) 8-12. 8 R.W. Biles, T.H. Connor, N.M. Trieff and M.S. Legator, The influence of contaminants on the mutagenic activity of dibromochloropropane (DBCP), J. Environ. Pathol. Toxicol., 2 (1978) 301-312. 9 S.J. Stolzenberg and C.H. Hine, Mutagenicity of halogenated and oxygenated three-carbon compounds, J. Toxicol. Environ. Health, 5 (1979) 1149-1158. 10 D. Whorton, R.M. Krauss, S. Marshall and T.H. Milby, Infertility in male pesticide workers, Lancet, 2 (1977) 1259-1261. 11 C.G. Biava, E.A. Smuckler and D. Whorton, The testicular morphology of individuals exposed to dibromochloropropane, Exp. Mol. Pathol., 29 (1978) 448-458.
108 12 D. Leroith, G. Potashnik, J. Dunn and I.M. Spitz, The exaggerated prolactin response to thyro1,2-dibromo-3-chloropropane-induced tropin-releasing hormone and metoclopramide in azoospermia, J. Clin. Endocrinol. Metab., 52 (1981) 38-41. 13 A.H. Conney and J.J. Burns, Metabolic interactions among environmental chemicals and drugs, Science, 178 (1972) 576-586. 14 D.E. Moody, B. Head and E.A. Smuckler, Reduction in hepatic microsomal cytochromes P-450 and b5 in rats exposed to 1,2-dibromo-3-chloropropane and carbon tetrachloride: Enhancement of effect by pretreatment with phenobarbital, J. Environ. Pathol. Toxicol., 3 (1979) 177-190. 15 D.E. Moody, G.A. Clawson, C.H. Woo and E.A. Smuckler, Cytochrome P-450 lowering effect of 1,2-dibromo-3-chloropropane and carbon tetrachloride, Fed. Proc., 40 (1981) 699 (Abst). 16 P.J. Tofilon, R.P. Clement and W.N. Piper. Inhibition of the biosynthesis of rat testicular heme by 1,2-dibromo-3-chloropropane, Biochem. Pharmacol., 29 (1980) 2563-2566. 17 E.A. Smuckler and E.A. Barker, Effects of drugs on amino acid incorporation in the liver, Proc. Eur. Sot. Study Drug Toxic., 7 (1966) 83-112. 18 E. Farber, Biochemical pathology, Annu. Rev. Pharmacol., 11 (1971) 71-96. 19 J.R. Gillette, J.R. Mitchell and B.B. Brodie, Biochemical mechanisms of drug toxicity, Annu. Rev. Pharmacol., 14 (1974) 271-288. 20 D.E. Moody, J.A. James, G.A. Clawson and E.A. Smuckler, Correlations among the changes in hepatic microsomal components after intoxication with alkyl halides, Mol. Pharmacol., 20 (1981) 685-693. 21 P.D. Sadowski and J.W. Steiner, Electron microscopical and biochemical characteristics of nuclei and nucleoli isolated from rat liver, J. Cell Biol., 37 (1968) 147-161. 22 G. Ceriotti, Determination of nucleic acids in animal tissues, J. Biol. Chem., 214 (1955) 59-70. 23 K. Burton, A study of the conditions and mechanism of thediphenylamine reaction for calorimetric estimation of deoxyribonucleic acid, Biochem. J., 62 (1956) 315-323. 24 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 25 E.A. Smuckler, O.A. Iseri and E.P. Benditt, Studies on carbon tetrachloride intoxication, I. The effect of carbon tetrachloride on incorporation of labeled amino acids into plasma proteins, Biochem. Biophys. Res. Commun., 5 (1961) 270-275. 26 D.E. Moody and E.A. Smuckler, Decrease in hepatic microsomal cytochromes P-450 and b5 after treatment with 1,2-dibromo-3-chloropropane (DBCP), Effect on synthesis of protein and heme moieties, Fed. Proc., 38 (1979) 845 (Abst).
101
THE INTEGRITY OF LIVER PROTEIN SYNTHESIS IN MALE RATS TREATED WITH 1,2-DIBROMO-3-CHLOROPROPANE
D.E. MOODY, GA. CLAWSON and E.A. SMUCKLER Department
of Pathology,
University of California,
School of Medicine,
San Francisco,
CA 94143
(U.S.A.)
(Received January 7th, 1982) (Accepted March 2nd, 1982)
SUMMARY Male rats treated with a single dose of 1,2-dibromo-3-chloropropane (DBCP) were tested for their ability to carry out the synthesis of liver proteins. In animals treated for 12 h, we found no changes in the uptake of [14C]orotic acid into liver RNA or the uptake of [3H]leucine into liver or serum protein. Uptake of [jH]leucine into the soluble fraction of the enlarged liver increased in proportion to liver size, while the uptake of [14C]orotic acid was unchanged. Examination of the ultrastructure of liver cells from rats treated for 12, 24, or 48 h revealed that the structure of the rough and smooth endoplasmic reticulum (RER; SER) were modified. An absence of ordered stacks of the RER and the presence of tangled nets of SER were noted.
INTRODUCTION
In test animals, acute exposure to DBCP has been found to result in damage to testes, kidney, lung, and liver [l, 21. Chronic exposure produces tumors in forestomach, mammary gland, lung, and nasopharynx [3-51, and effects the reproductive capacity of male animals [6]. In addition, DBCP produced mutagenesis in bacterial systems [7-g]. The reproductive lesion has also been found in men occupationally exposed to this compound [lo, 111. The mechanism of the reproductive failure is as yet unknown; an alteration in steroid metabolism is a plausible explanation [ 121. The cytochrome P-UO-dependent microsomal mixed-function oxidases have been
Abbreviations: DBCP, 1,2-dibromo-3-chloropropane; ER, endoplasmic reticulum; RER, endoplasmic reticulum; SER, smooth endoplasmic reticulum; TCA, trichloroacetic acid. 0378-4274/82/0000-0000/$02.75
0 Elsevier Biomedical Press
rough
102
found to play a role in many facets of steroid metabolism including their synthesis and catabolism [13]. Whether alterations in the activity of this system are reflected in alterations of steroid function has not been thoroughly examined. We have recently found that a single treatment with DBCP leads to a decrease in hepatic cytochrome P-450 [14] and have presented preliminary evidence that this decrease occurs in microsomes from testis, lung, kidney, and intestinal mucosa [15]. The decrease in testicular microsomal cytochrome P-450 has also been reported by Tofilon and coworkers [16]. These authors also found an alteration in the synthesis of heme in testicular tissue. One manner in which toxins can cause a decrease in protein levels is through a reduction in protein synthesis, either at the level of transcription (RNA synthesis) or translation (protein synthesis). Several toxins are known to have gross effects on these cellular actions [17-191. In the current study, we have tested the effect of DBCP on several parameters of protein synthesis in liver cells. We found that DBCP had little or no effect on the overall process of protein synthesis. These findings, and previous findings on the response of microsomal components to alkyl halides [20], suggest that the action of DBCP on microsomal cytochrome P-450 is specific and not the result of a major lesion in the hepatocyte. METHODS
Male Sprague-Dawley rats (caesarian delivered, Charles River, Wilmington, MA) were housed in pairs in steel screen-bottomed cages with food and water provided ad lib. Food was removed 18 h prior to killing. DBCP (technical grade, Occidental Chemical Co., Lathrop, CA) was dissolved in mineral oil (2% v/v) and 0.1 ml/kg administered by oral gavage. Control rats received an equal volume of mineral oil (5.0 ml/kg). 2 h prior to killing, either 250 &i/kg (4, 5)-[3H]leucine (New England Nuclear, 60 Ci/mmol) or 30 &i/kg (6)-[14C]orotic acid (New England Nuclear, 28 mCi/mmol) were injected via the tail vein. At the times specified, animals were anesthetized under light ether, blood was collected by cardiac puncture, the livers were perfused with 0.9% saline, removed into tared beakers, minced, weighed, and homogenized in 3.4 ~01s. of 0.1 M potassium phosphate (pH 7.4). An aliquot of the homogenate was precipitated with an equal volume of 20% TCA. The supernatant was collected for determination of the unincorporated label taken up by the liver. The pellet was then extracted as described by Sadowski and Steiner (211, which first removes lipids and then successively extracts RNA, DNA, and protein. Aliquots from the initial TCA supernate and the latter extracts were dissolved in protosol, suspended in aquasol, and the radioactivity was determined by liquid scintillation counting. The contents of RNA [22], DNA [23], and protein [24] in the respective extracts were determined by the methods cited. Sections were taken from livers after perfusion and fixed in 1% osmium tetroxide in S-collidine buffer (pH 7.4) prior to preparation for examination by electron
103
microscopy. Serum was prepared from the blood samples and separated into albumin and globulin fractions as previously described by extraction of albumin from TCA precipitates of serum with methanol [25]. RESULTS
The livers of male rats increase in weight following treatment with DBCP [14]. Relative liver weights were 1.28 times greater than control livers after 12 h of treatment (Table I). The content of total liver DNA did not change signi~cantly following treatment, but liver RNA and protein increased in a parallel manner to the liver size. These results suggest that the increase in liver size arises predominantly from cellular hypertrophy rather than cell division. The uptake of the radioactively labeled precursors into the liver was estimated from the radioactivity recovered in the supernates after precipitation of the liver TABLE I THE EFFECT OF 12-h TREATMENT WITH DBCP ONLY LIVER SIZE, DNA, AND RNA, AND LIVER AND SERUM PROTEIN CONTENT AND THE INCORPORATION OF LABELED PRECURSORS INTO EXTRACED RNA AND PROTEINa Sample Liver weight g/100 g body wt TCA supernate [t*C]CPM/lOO g body weight (lO-6) [3H]CPM/i~ g body weight DNA extract mg/lOO g body weight RNA extract mg/lOO g body weight [‘%]CPM/pg RNA Protein extract mg/lGil g body weight [sH]CPM/mg protein Serum albumin (mg/ml) [sH]CPM/mg protein Serum globulin (mg/ml) [sH]CPM/mg protein
Control
DBCP
3.55 +
0.24
4.56 +z 0.25b
1.19* 78.6 +
0.17 9.5
1.19 f 0.23~ 93.5 + 17.4”
6.56 +
0.8
10.5 f. 18.1 rf
1.9 6.6
368 310.8 2.19 979 7.20 816
f 78 f 33.3 zk 0.58 + 267 + 0.59 t 156
7.09 t
1.18c
12.1 + 0.4” 15.7 + 4.4 468 f 6b 322.3 zk 34.3 2.32 zk 0.39 984 f 80 7.70& 1.43 789 f 86
aExtracts of liver homogenates and serum were prepared from rats Iabeled with [3H]leucine ([sH]CPM) or [tK]orotic acid ([t4c]CPM), as described under METHODS. Values are the mean + SD for at least 4 animals. Due to the change in liver size after treatment with DBCP, the values were routinely expressed per g liver and per total liver of a 100-g rat. Only the latter values are presented, but significant differences are noted for both cases. bsignificantly different from controls when values are calculated for total liver/l00 g body weight, P-20.05. %SigniBcantly different from control when values calculated/g liver, P < 0.05.
104
homogenates with TCA. While the uptake of [i4C]orotic acid into the total liver was not significantly changed (Table I), the uptake of t3H]leucine increased along with the increase in liver size (Table I). The mechanism of entry of these two compounds may, therefore, respond differently to treatment with DBCP. Liver RNA and protein were extracted from TCA precipitates of liver homogenates and the specific radioactivity of the two fractions was determined. In animals prelabeled with [14C]orotic acid or [3H]leucine no significant change was found in their incorporation into extracted RNA or protein, respectively (Table I). Furthermore, the incorporation of label into serum-albumin fraction, a protein synthesized and secreted from the liver, and the serum-globulin fraction, proteins of a heterogeneous origin, were not affected. The synthesis of RNA and protein in livers of rats does not appear to be effected 12 h after treatment with DBCP. Sections of livers from rats treated for 12,24, and 48 h with DBCP were examined by light and electron microscopy. The livers of our control rats were not different from previous descriptions of Sprague-Dawley male rats. At 12, 24, or 48 h following DBCP the only consistent light microscopic finding was a dispersal of the cytoplasmic basophilia. Ultrastructural examination revealed that the ER was modified (Fig. 1). Stacked arrays of RER were almost absent. Ribosomes remained attached to the membrane and whorls and aggregates of cytosolic polysomes were detected. Dilation of the cisternae of both the RER and SER was also apparent, and areas of the SER were aggregated in tangled nets (Fig. 1). In addition, the hepatocytes from animals tested with DBCP contained a number of non membranebound lipid droplets. It was observed that the mitochondrial cristae did not always penetrate to the middle of the organelle when compared to controls. The plasmalemma, nuclear envelope, nucleoli, and other organelles, were not different from controls. DISCUSSION
Several reports on the acute and chronic effects of DBCP on male rats have mentioned alterations in liver cells which include liver enlargement, congestion, and focal centrilobular necrosis [l-5]. In previous studies we have noted that treatment with DBCP causes a decrease in hepatic microsomal cytochromes [14]. This is associated with a change in the relative content of microsomal fatty acids, particularly arachidonic and linoleic acids [20]. Several compounds which also cause a decrease in hepatic microsomal cytochrome P-450 cause a decrease in the synthesis of hepatic proteins [17-191. This latter defect may in part be responsible for the decline in cytochrome content. The results of the current study suggest that DBCP does not effect an overall decrease in protein synthesis either by a direct action on the incorporation of amino acids or indirectly, through an effect on the synthesis of RNA. These studies, however, cannot rule out the action of DBCP on the synthesis of specific messengers
Fig. 1. Electron micrographs of sections of liver tissue taken from (A) control male rat or (B) a male rat 12 h after treatment with 0.1 ml/kg DBCP. The photos demonstrate the difference in the: spaltial arrangement of the RER, which is more commonly dispersed in the treated animals, and the pre sencle of tangled nets of SER. Lipid droplets (Id) are present in hepatocytes from the treated rats. Notice that the other organelles are not markedly effected by the treatment. The bar represents 1 pm.
(i.e., the messenger for cytochrome P-450 apoprotein) or the regulation of its translation. Another point of regulation for the synthesis of microsomal cytochromes is the turnover of the heme moiety. Tofilon and coworkers [16] have described a decrease in the uptake of the heme precursor, b-aminolevulinic acid, into testicular microsomal heme. This was associated with a decrease in cytochrome P-450 and changes in certain heme synthesis enzymes. Preliminary results from our
107
laboratory show that a similar defect in heme synthesis may occur in liver tissue, as a 15% decrease in the uptake of d-aminolevulinic acid was found after 12 h of treatment 1261. At this point the importance of the decrease in cytochrome P-450 to the toxic effects of DBCP are not known. Due to the role of cytochrome P-450 in the metabolism of numerous endogenous lipids, including steroid hormones, the decrease may be linked to subsequent physiological changes which follow exposure to DBCP. As DBCP and other alkyl halides, which also effect cytochrome P-450 levels [20], are known to effect the reproductive capacity of test animals and humans [9-l 11, this may prove to be an important step in the toxic action of these compounds. ACKNOWLEDGEMENT
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