Document 5DnYyJmqz0Zk3rww5BnQ0JYZz

Rough Draft Translation INDUCTION OF MICROSOMAL LIVER ENZYMES AFTER POLYCHLORINATED BIPHENYLS (PCB) AND FOLLOWING STRESS By II. F. Benthe, A. Schmoldt, and H. Schmidt Arch. ToxlKoI. 29, 9T-106 (1972) , Translated by Michael Dub, March 12, 1975 ' ABSTRACT One i.p Injection of PCB induces a high stimulation of rat liver microsomal drug oxidising enzymes; equimolar doses of tctrAchlorobiphenyls are distinctly more effective than dlchloroblphenyls. The effect can still be demonstrated distinctly four veeks after application. During this time j the liver concentration of tetrachlorobiphenyls decreased to 7ug/g tissue whereas the concentration of adipose tissue rose to '?CP uc/n. Mw stress tctrcchlor&biimenyla at* mobi lized along with fat, causing an increase of liver concen tration to 52 Ug/g. Simultaneously, a new stimulation of microsomal activity can be seen, nearly equal to the maximum stimulation ^ days after l.p. application of 500 mg/kg b.w. Parallel to microsomal stimulation there is an increase of relative liver weight Stress induced activation of microsomal activity caused by PCB redistribution Is important for the evaluation of PCB in environmental toxicology, in view of rising PCB concentra tions in human adipose tissue. Besides DDT and its metabolites, polychlorinated biphenyls (PCB#s) in an increasing extent are found in marine food chains. In feeds, birds, in human adipose tissue. 1 HONS 066832 and in human milk (Kocmann et al., 19^9 Holmes et al., 1967; Rlscbrough et al., 1969; Biros ct al., 1970). As far as the few qualitative analyses show. It is 11 matter of higher chlorinated ?CB`'b (penta- to hepta-chlorobiphenyI9) (Duke ct al., 1971; Bagley et al., 1970). Experiments on animals with these compounds showed that a strong Induction of microsomal enzymes could be induced (Risebrough et al., 1969)* Since at present attempts have been made to replace the higher chlorinated PCb's by lower chlorinated compounds, the toxicological properties of these sub stances (di- to tetrachlorobiphenyls) are of a particular interest. In doing this, two exposure forma should be distinguished: the chronical absorption in the organism via a general environmental pollution with these compounds and an accute resorp tion of relatively large quantities through the coimnercial application of PCb's, among others as - insulation, heat exchange, and hydraulic fluids. Having described the distribution of the coimterclal PCB mixture Pydraul a 200 (Monsanto) In the organisms of rats (Benthe ot al., 1972) and having established an acute microsomal enzyme Induction (Nlssen, 1970), now wa should investigate the. Lra>.Juialluu vl u> wuoyuie induction and the possible mobilization of g totrachlorobiphenyl Aroclor 1248 (Monsanto) deposited in the adipose tissue. METHODS Male Wiatar rata (200 to 230 g) obtained a single intraperitoneal injection of tetrachlorobiphenyl (Aroclor 1248) and equimolar doses of dichlorobiphenyl (Aroclor 1232), respectively, (in the following abbreviated as TCB and DCB, respectively), in 1 + 1 mixture with olive oil, and until their killing obtained food (Altromin-R) and water in unlimited quantities. Rats of the same strain which obtained Intraperltoneal injections of olive oil only serve as controls. The test animals were killed after the indicated periods of time (between 3 PM and 6 TM) by a blow in the neck and decapitation. Tho conditions of temperature of 3*C. stress consisted 0$ withdrawal of food and of a surrounding -2- MONS 066833 Mlcrosome Preparation Iirancdlately after killing the animals, the livers were removed and freed from blood in an ice-cold 0-1 m phosphate buffer of pH 7-^, were weighed, were chopped into small pieces in the ice-cold phosphate buffer of pH 7`^ were rinsed, and then were homogenized (Potter-Elvehjem) in 8 volumes of che fresh buffer. The homo genate was centrifuged for 10 minutes at 30 xg and 20 minutes at 17,000 xg, the clear supernatant was siphoned off and was centrifuged 60 minutes at 105,000 xg (Rotor Splnco L2). The deposited microsomes, after decanting the supernatant and after rinsing the loosely packed upper layers with a fresh phosphate buffer while leaving the glycogen sediment, were rehomogenized and centrifuged again during 60 minutes at 105*000 xg. The microsomes washed in this manner were taken up in the fresh buffer In such a way that about Uo mg protein/ml were suspended. The oxidative O-demethylation of p-nltroanisole, measured according to the Hotter U9&3) method, served as a measure of the activity of the microsomal enzymes. In these tests, 2 ml incubation batch contained 200 umol phosphate buffer of pH 7.4, 5>5 Mrool glucose-6-phosphate (Boehringer), 12.5 Uol nicotinic acid amide, 30 umol MgCl2*6 H20, 0.5 umol p-nltroanisole, 2 mg microsomal protein, 8C0 mU glucose-6phosphate dehydrogenase. The incubation batch was saturated with 02 and was filled Into a 1 cm cell. After about 3 minutes preincubation at 37*C, the reaction was started with the addition of 0.02 umol NADP. The formation of p-nitrophenol was registered by the extinction increase at 1*20 nm in spectrophotometer PM Q? (Zeiss) against a reference cell (without NADP) in the time intervals of 1 minute over a period of 15 minutes. The reaction proceeded to about the 10th minute with a constant rate. For the microsomes of the untreated normal animals the p-nltroanisolc conversion amounted to C.72 nmol/mg microsomal protein/10 min. at 57*C. This value was verified in each series by means of 2 control animals, was set 100%, and the enzyme activity of the rats treated with tetrachlorodiphcnyl was referred to it. MUN5 066834 -5 - Analysis The determination o the protein contents of the microsome preparations was carried out by the biuret reaction with bovine albumin as a standard substance. To deter mine the PCB contents in the liver, 1 g of the tissue was ground with anhydrous NapSOn to a dry powder, the powder was extracted twice with 4o ml CHC13 (analytical grade), the combined CMC13 extracts were evaporated In a rotary < evaporator to dryness, and the residue (in the following designated as total lipidc) was deter mined gravimetrically. The extraction of PCB from the total llpide was carried out according to Grant et el. (1971), and the quantitative determination was accomplished according to Benthe et al. (1972). By this method, the yield amounted to 85 + 5.1%. All the chemicals were of "analytical grade, hexane and heptane were spectrally pure (Uvasol, Merck), G-6-P, G-6-PDH, NAD7 were obtained from Bohringer Co., p-nitro- anisole, analytical grade, from Schuchardt. The donation of tetrachlorodlphenyl RR Aroclor 1240 and of dlchlorobiphenyl Aroclor 1232 by Monsanto Is acknowledged. RESULTS To find a suitable dose, PCB was administered to the rats In increasing quantities, and the microsomal activity increase was measured after 2 and 4 days, respectively (Table 1). According to these tests the threshold dose is about 5 mg TCB/kg,while that for DCB la considerably higher. In further studies of TCB, to expect a definite microsomal activity Increase, a dose of 500 mg TCB/kg was chosen. As Table 1 shows, an equimolar dose of DCB has a considerably weaker effect. -4 - MGNS 066635 Table 1. Increase! of the Oxidative Domethylaclon as a Function of Equimolar Quantities of TCB and DCB; Average Values from U Aniraals ~~ 9 wb PCD/hg Days After Injection Microsomal p-Nitroanisole Demethylation 7 of Control _ Control Control 5 TCB 5 TCB 25 TCB 50 TCB 500 TCB 58.2 DCB 382 DCB 382 DCB 2 100 + 4.6 k 100 + it.6 2 l46 + 65.O k 108 + 51.2 k 146 + 55.6 k 208 + 20.0 k 488 + 14.0 k 146 + 25-0 2 515 + 54.4 k 266 + 52-7 *1007. 6-72 + O.5I nmol p-nltrophenol/mg microsomal protein/10 min. Figure 1. Demethylation of p-nltroanlsole in percent of the control (1007. > 6.72 nmol + 0.51 nmol p-nltrophenol/mg microsomal protein/10 min) as a function of time after a single l.p. injection of 500 mg TCB (upper curve) and 582 mg DCB/kg KC (lower curve), respectively. Average values from 5 rats + s. A test of the transient course of the Induction effect shows that the maximum is reached 3 to 4 days after 500 mg TCB/kg, up to the 7th day a rapid decrease of the activity takes place, and after that it hardly decreases and consequently, even 20 days after the TCB application,the microsomal activity exceeds the control value by moru than Lwo-fold. Individual measurements 8 and 13 weeks after application still -5- MOMS 066836 always gave Increased values. On the other hand, relative to TCB, the extent of activation after an equimolar dose of DCB Is considerably lower, and the maximum seems to be reached after 2 days. However, the effect is still clearly detectable after lH days (Figure l). Figure 2. The relative liver weight (g/100 g body weight) In percent of the control aa a function of time after a single l.p. injection of 500 mg TCB/kg body weight. Average values from 6 rats (the value after 55 days from k rats) + s. Figure 5* The TCB concentration of the liver and the microsomal p-nitroanisole demethylation (in % of the control) as a function of time after a single dose of 500 mg TCB/kg body weight (see Figure 1) and afterthe 28th day of the stress duration. For every 6 control animals 100'i 6.72 + 1.5 (20ch day), I5.2 + 2.15 (5^3t day), 10.2 + 1.2 (5lith day) nmol p-nitrophenolAng microsomal protein/10 min. -6- MQNS 066837 An increase of the liver weight is parallel to the activity increase (Figure 2). A steep weight Increase within the first 5 days is followed by a slow decrease, and only after h weeks the liver weight approaches that of the controls. Figure 1*. The TCB accumulation in the liver under stress conditions. The upper curve: TCB concentration at the beginning and after 3 and 6 days stress plotted against the corresponding liver weights. The lower curve: con version of the TCB concentration at the stress start (26 days after the TCB application) to the decreasing liver weights. Figure 5* The TCB quantity (ug TCB) in the total retroperitoneal adipose tissue (shaded column) and the TCB concentration (ug/g adipose tissue) after o single injection of 500 mg TCB/kg body weight and after the following the 3 day long stress. Average values from 6 rats + sx. For the PC8 concentration in the liver (Figure 3) o maximum value (150 TCB/g) is measured 2^ hour9 after the application which then decreases within 7 days in about exponential course to a value of 22 ug/g, after which it still decreases only slightly. A comparison of the TCB contents with the microsomal stimulation shows that the stimu lation proceeds parallel to the PC2 content In the liver with a time phase shift of 2 to 3 days. -7 - MQNS 066838 It 1b known from previous distribution studies (Benthe et al., 1972) that PCB is redistributed in the adipose tissue. In the present investigation the PCB concen tration amounted to 252 U-g/g adipose tissue 28 days after application. Now the question arises whether under stress conditions the PCB stored in the adipose tissue is mobilized and triggers a renewed Increase of the enzyme activity in the liver. For this purpose, 28 dayi^f^e PCB application the rats were exposed to a surrounding temperature of 3*C and they were deprived of food. Figure 6. The course of the relative liver weight (g/100 g body weight) of the TCB treated rata (upper curve) end of the untreated control animals under the stress conditions. The starting value four weele after a single l.p. injection of 300 mg TCB/kg body weight. ForTanimal groups after 3 and 6 days the following values were determined: O-demethylatlon, TCB concentration of the liver and of the adipose tissue and the relative liver weight. As a control, we used under the same conditions the control animals of the same rat group which were treated with olive oil. As Figure 3 shows, the TCB concentration in the liver Increased more than two-fold after 3 days stress (from 7*0 to 10.3 Ug/g), and to 3? Mtg/g after 6 days stress. This concentration Increase cannot be explained by the decrease in the absolute liver weight. When wc plot the PCB starting concentration as a function of the liver weight, we obtain the lower curve of Figure U. The actually measured concentrations correspond to the -8 - HOMS 066839 upper curve and show an extremely steep increase. This increase is based on a mobilisation of PCD from the adipose tissue. Figure 5 shows that under stress the TCD quantity in the entire adipose tissue decreases, but the TCB concentration (Ufi/g fat) remains constant due to lipolysis. After 6 days of stress, the entire retroperitoneal adipose tissue dissipated so that no measurement could be carried out anymore. With the increase of the TCB concentration in the liver, a renewed microsomal stimu lation takes place, which after 6 days of stress amounts to ^^4% and, therewith, reaches again almost!the maximum of the initial stimulation after 500 mg TCB/kg. The weight increase of the liver found at the beginning is measurable again under the stress conditions (Figure 6): while the relative liver weight of the control group abruptly decreases in the first 5 days, it shows a clear tendency toward An Increase in the animals treated with TCB. DISCUSSION In the investigations published to date on the Induction of microsomal liver enzymes in rabbits (Vllleneuve, 1971)> rats (street, cited from Peakall and Linear, 1970) and falcons (Lincer and Peakall, 1970) with higher chlorinated biphenyls (penta- to hepta-chlorobiphenyls) singular observations were always made at the end of the chronical feeding tests. Nissen (1970) was, however, able to show that even a single short-lasting exposure caused this induction. As Table l and Figure 1 show, low chlorinated biphenyls after a single application lead also to a microsomal stimulation. Now it was of interest to ask how rapidly TCB Is eliminated from the liver and how long, in relation to it, the increased activity of microsomal enzymes remains. WhLle from the classical Inducer pheno barbetal, for example, the microsomal aminopyrine-demethylase is again found normal ) days after the last Injection (Orrenius and Ernster, 19& ) the increased enzyme -9 - MQNS 0666*0 activity after DDT lasts for several weeks (Chazal et al., I96*4) Consequently, the activity of TCB can be very well compared with that of DDT: k weeks after the TCB administration, the activity of p-nitroanisole demethylase Is decreased to **77. of the maximum value and still remains exactly as high as after 2 weeks. Individual measurements after 6 to 13 weeks still did not give a normal value. The TCB con tents of the liver decreases rapidly within the first 3 to 5 days, but after that only very slowly. After 4 weeks there are still 5?* of the initial value present In the liver (Figure 3)* This finding is In good agreement with the analyses of Grant et al. (1971)/ who 3 weeks after administration of 300 mg pentachlorobiphenyl (Aroclor 125*0/kg still found I3Z of the concentration of the 2nd day. Likewise, the Increase of the relative liver weight by Aroclor 125*1 is also valid for TCB (Aroclor 12**8). The transient course (Figure 2) is parallel with the microsomal activity and reaches?maximum, the double weight of the controljwlth 6.757 (of the body weight). Besides, it is questionable whether this enornmous Increase can bu explained only by the increase of the endoplasmic reticulum. When we compare the transient course of the TCD contents of the liver with that of the adipose tissue, it becomes clear that a large part of TCB Is redistributed from the liver into the adipose tissue, as we were also able to detect after the FCU application ss aerosol (Benthe et al., 1972). Now v raise the question: is the TCB deposited In the adipose tissue tnoblllzablc when the adipose tissue is dissipatedunder lipolytic conditions? For DDT this redistribution from the adipose tissue was reported many times (Dale et al., 19&2; Brown, 1970; Findlay and Freltag, 1971)* Moreover, when 7 ug TCB/g liver are still sufficient for maintaining the microsomal activity on a higher level, then in the case of a redistribution a repeated increase of the induction could be expected under the condition that the TCB can act sufficiently long on the liver. Figures b and 5 show a confirmation of our hypothesis that during dissipation of the adipose tissue TCB is mobilized and the liver concentration increases again. 10 - MGNS 066841 The attained concentration with 32 ug/g liver tissue corresponds to about the T day value after an l.p. injection. The increase of the O-denethylation after 3 days stress with nearly 300?* corresponds to the 7 day value and after 6 days reaches again almost the maximum value. The increases refer to the untreated control Animals of the same colony which were exposed to the same conditions. This is also therefore important since the conditions of fasting alone lead to an increase in the activity of the microsomal enzymes (Figure 3)/ as was already shown by Kato (1967) and Cram et al. (1970). The increase of the relative liver weight also agrees with these findings. In addition, it should be noted that the decrease of the absolute body weight in the groups showed no significant differences. Therewith, it can be considered as proved that under the lipolysis conditions a concentration of 260 ng TCB/g adipose tissue is sufficient for inducing micro* ooinal enzymes of the rat liver. Although this concentration is bO times higher than in the adipose tissue of a normal person (6 ppm) (Quentin, 1972), but in view of Ilia long retention in the adipose tissue in an occupational exposure,it con easily lead to an accumulation up to these concentration ranges. / * 11 MONS QbbQkZ REFERENCES lUglry, 0. E., Kcirbel, IV. L, Cromartic, K. s Menlilintlion of polychlorinated biphenyls in two hold eagles by combined 3i)irpiid chromatography moM portfttmftry. J. Am. Oflie. Ami. flirm. .VI, 201 (11)70). - Henthr, H. ., Knop, J., Sehmnldt. A.: Aufnahme und Verteihmg naelt Inhalation polychlomtter lhphsnyl* (PCD). Arth. Toaikol 23, Bft--5 (1072). TJiros. K. .1., Walker, A. C.. Mrdbary, A.: PCD'* n h'tman itlipoM tissue. Rull. Environ, f'onlnni. 'i'liiod, . .*!7I , 1 Brouii. .1. ft.; The effort of environment mid dietary slrraa on tlio mncrnlrntron of . DDT in rot". Tnsh-ol. appl. VhMmavnI. 17, 5>M (1!)71>). ; Dale, W. K-. Caines. T. H., ilnyc, W..1.: .Ktnrago and excretion of DDT in starred j rata. Toxieol. nppl. Pharmacol. 4. HO (1002). Duke. T. IV., Uwe, Wilson, A. !., Jr.: A polyehbwinntcd Diphenyl (A/oeMor 12.74) in the water. sediment ami biota of Esramhia Hoy. Florida. Hull. Environ. [ Contain. Toxicol. 'i, 17) (1071). 1 Findlay, 0. M.. I'Vrilng. A. S. IV. de.t DDT movement from adipocyte to tnuaclocrU during lipid ulilizatioo. Nature (Und.) 220,Off (1971). ( Ohara), A.. Kornnshy, W.f Portie, J., Klrmpau. J.: llcsrhletiniouns von Kntjjif- ' (nngsrraktionrn 'lurch vcrvrhtcdcue Iiwcklixide. Noimyn-Sehmiedebcrg* Arch, j Phnrmak. 24!l. 1 (t!H4). 1 Cram.T. E.. Gnnrinn. A. M.. Srhnniw. D. II., Daria, 1). C., Reagan, It. 7,., Gilctte, ! J. 11.: The effect of atarration on tiia kinetics of drag oxidation by hepatio | mlerosomnl cnr.ymea from male and female rata. J. Pharmacol, exp. Ther. 174, i 13(1970). { Grant, D. h, Phillips, IV. R., Villcnmtve, D. C.: Metabolism of a imlvehlorinated . Diphenyl (Aroehlor 12:71) mixture in the rot. Hull environ. Contain. Toxicol. [ *,103(7971). I llolmrs. D. C., Simmon*,,). 77., Tat ton, 0. C.: Chlorinated hydrocarboos in British ' wildlife. Nature (Lend.) 21ft, 327 (1907). j Kato, H.: Effects of starrntion and refeeding on the oxidation of drugs by User : ynicrosomea. H*ochcni. Pharmacol. Ift, S7I (19D7). 1 Eci7,;r>,. ., NWvi. lie Braun. M. 1`. trn, Vos, H. H. do: Chlorinated Diphenyls In Osh, mussels and birds from rircr Rhine and the Netherlands roastal area. Nature (Und.) 221, 112ft (1909). Orrrnius, S., Ernatcr, L.: I'heuobatUdal induoeit avnlhesis of the oxidative dome* ; thylaliny eniymea of rat liver mhrotwrmos. Hiochcm. biophys. lies. Commun. 1C, Oft (tool). I Katler, K. j.: Untersurhiingen fiber die Hcmmung des oxldativen und hyilmlyti* chen Arincimiltclsloffwfchsels. Hahditation. Hamburg 1963. , NImco, K.: Dtr EinQuO ehtorierter Diphony)o aiif die Uberfunktion von Ratten. > 1Dissertation, Hamburg 970. ' j Paakalt, D. B., Linrcr, J, C.: I'olythlorinsled Biphenyls. Another long life wide ; spread chemtenl in the en\ iromnent. Biol. Sri. 2ft, 903 (1970). j Quentin, K. E.: Pesticide im IVasscr -- Dcstimmung, Entfemuog und Grant* 1 wertc. Vortrng, Hamburg 1072. ; Riatbrough, R, IV., Ricche, P., IVakatt, D. It., Hermann, S. G., Kirveo, M. N.: : PCD in the global Ecosystems. Nalurn (Uml.) 22ft, 1096 (1903). 1 THIeneuve, D. C., Grant, D.),., Phillips, E. J., Dark. M. L., Cle?g. O. J.: Effect* of PCD administration on mirrosnmsl enzyme activity in pregnant rabbits. Bull. Environ, Contain. Toxlool. *, 120 (1971). 12 - MCNS 066643