Document 6B00ML89DarRJrgo4eLEVgxR1

Toxicology, 5 (1975) 69-77 Elsevicr/Noith Holland, Amsterdam - Printed in The Netherlands POTENTIATION OF CARBON TETRACHLORIDE 11EPATOTOXICITY ]N RATS BY PRETREATMENT WITH POLYCHLORINATED BIPHENYLS GARY P. CARLSON Department of Pharmacology and Toxicology, College of Pharmacy, University of Rhode Island, Kingston, li.l. 02881 (U.S.A.) (Received March 3 Gib, 197 5) (Accepted May 6th, 1975) SUMMARY Pretreatment of male rats with Aroclor 1254 at a dose of 25 mg/kg i.p. for 6 days resulted in potentiation of the hepatotoxicity of inhaled carbon tetrachloride (CC1) as evidenced by a decrease in liver glucose-6-phosphalase and elevations of serum glutamic oxalacetic transaminase (SGOT), se rum glutamic pyruvic transaminase (SGPT), isocitrate dehydrogenase, and sorbitol dehydrogenase. Aroclor 1254 alone did not demonstrate hepatotox icity. Aroclor 1264 administration resulted in large increases in cytochrome c reductase, cytochrome P-450 (448) and p-nitroanisole demethylation. Sub sequent exposure to CC14 vapor resulted in over 70% decreases in the latter two parameters. The potentiation was dose-dependent with a dose of 5 mg/kg or higher being effective. Aroclor 1260 administration gave results similar to those of Aroclor 1254, but Aroclor 1221 enhanced CC14 toxicity to a lesser extent. INTRODUCTION Since McLean and McLean [1] first demonstrated that the enzyme-induc ing agent DDT was capable of potentiating the toxicity of CC)4> many investigators have found this to be true of other enzyme-inducing agents such as phenobarbetal [2--4). In addition to phenobarbital and DDT, other investigators have looked at the influence of the polycyclic hydrocarbon type inducers which differ in the microsomal carbon monoxide binding pig ment induced, i.e. P-448 rather than P-450, and in the spectrum of enzymes induced [6] Pitchumoni et al. [6] demonstrated that benzo[a]pyrene was similar to phenobarbital in enhancing the toxicity of CC14. However, Suarez Abbreviations1. DDT, 1,1 ,l-trichloro-2,2-bis(pclilorophonyl)ethanc; PCB, polychlorinated biphenyl; SOOT, serum glutamic oxalacetic transaminase; SGl'T, serum glutamic pyruvic transaminase. 69 DSW 026463 et al. [3] demonstrated that 3-methylcholanthrene protected against the hepalotoxicity of CCI4, It thus appears that enhancement versus protection is not simply a matter of cytochrome P-450 versus P-448. Recently Alvaros and coworkers |7) have shown that the polychlorinated biphenyls are unique as enzyme-inducing agents in that they induce the formation of cytochrome P-448 as does 3-methylcholanthrene, but they resemble phenobarbita! in that they bring about a more general type of induction. In view of this uniqueness, it was of interest to ascertain what effects these compounds have on CCI4 toxicity. Although the PCB's as commercially used both in the past and in the present are complex mixtures, they are numbered according to the percent chlorine by weight, i.e., Aroclor 1254 contains 64% chlorine. Since the chlorine content appears to have an influence on the inducing potential of the PCB's [8,9], it was also important to determine if percent chlorine would be a factor in alteration of CC14 toxicity. METHODS Adult male albino rats (Charles River Breeding Laboratories) were used. They were allowed food and water ad lib. and were housed in temperatureand light-controlled rooms. They were injected i.p. with Aroclor 1254 dis solved in com oil at a dose of 25 mg/kg unless otherwise indicated, Controls received com oil alone. Injections were made daily for 6 days and 24 h after the last dose the rats were exposed to CC14 vapor in a dynamic inhalation chamber [10] for a period of 2 h. Chamber concentrations were determined U6ing a Packard gas chromatograph with a flame ionization detector. 22 h after exposure the rats were lightly anesthetized with ether, liver and tail vein blood samples taken and various parameters of hepatotoxicity were assessed. Liver and body weights were recorded. Liver glucose-6-phosphatase was measured using the procedure, of Harper [11] with rnaleate buffer, pH 6.25. p-Nitroanisole demethylation was determined according to the method of Netter and Seidel [12] as modified by Kinoshita et al. [13]. SGOT and SGPT transaminases were measured utilizing the method of Reitman and Frankel [14]. Isocitrate dehydrogenase was determined ..ccording to the procedure of Ellis and Goldberg [15] and sorbitol dehydrogenase by the method of Gerlach [16]. In the microsomal cytochrome experiments, the livers were perfused with cold isotonic KC1, removed, and homogenized -l cold isotonic KC1. The homogenate was centrifuged at 90V0 g for 20 min in a Sorvall Model RC2B refrigerated centrifuge and the supernatant furllxr centrifuged at 105 000 g for 1 h in an International Model IVGO Ultracentrifuge. Cytochrome c reduc tase activity and cytochrome P-450 content in the resulting microsomal fraction were measured according to the methods of Dallner [17]. Protein concentrations of the microsomes were determined by the method of Lowry et al. [18]. 70 0SW 026464 The value expressed is the mean i standard error. Student's t test was used to compare means at a chosen level of significance of P- 0.05. RESULTS The effects of exposing the rats to 3600 ppm of CC14 for 2 h on 3 parameters are presented in Table 1. Pretreatment with Aroclor 1254 re sulted in an increase in the liver to body weight ratio. This increase was still greater in those animals exposed to CC14, although CC14 exposure by itself did not alter the ratio. At this level of exposure there was a small decrease in liver glucose-6-phosphatase activity due to the CC14 alone. However, in those animals pretreated with Aroclor 1254 there was a large decrease in activity to one-third of the air-exposed level. As a further indication of damage to the functioning of the liver, drug metabolism was measured since this is known to be decreased by CC14 and the effect potentiated by phenobnrbilal but protected against by 3-methylcholanthrene [4]. As indicated in Table I, a decrease in activity was seen v/ith CC14 inhalation. Aroclor 1254 adminis tration resulted in an 8-fold increase in activity which decreased dramatically following CC14 inhalation. To further study the potentiation by Aroclor 1254 on the decrease in microsomal enzyme activity due to CCI4, animals were exposed to 4200 ppm of CC14 and cytochrome c reductase activity and cytochrome P-450 content were measured. As indicated in Table II, cytochrome c reductase activity was elevated 2-fold by Aroclor 1254. In neither the controls nor the Aroclor-pretreated rats was the uctivit.y altered by CC14. In the case of the P-460 (P-448) content, Aroclor 1254 pretreatment resulted in a 3-fold in crease in this cytochrome. CCI4 inhalation resulted in a decrease in both the TABLE I EFFECT OF AROCLOR 1264 AND CCI,, ON LIVER WE1GHT/BODY WEIGHT, GLUCOSE-G-P1IOSPHATASE, AND p-NJTKOANISOLE DEM ETHYL ATI ON Treatment N" Liver wt. y , 00 Glucose-6- p-Nitroanisole Body wl. phosphatase1* demi'ihylationc Corn oil--Air Corn oll-CCI4<s Aroclor 1264--air Aroclor 1264--CCI4 5 6 6 6 4.26 0.07 4.02 * 0.16 4.96 0.25* 6.911 0.1 3f- 12.0 1 0.66 10.2.1 0.58' 10.2 1 0.34 3.G i 0.37'-* 5.1 1 0.39 2.9 10.17' 43.1 1 0.42* 12.01 1.571* * Number of rata b amoleB P04/g/min. e *ig/60 mg/30 min. a 3600 ppm for 2 h. * 26 mg/kg i.p. for 6 days. 1 Significantly different (P < 0.06) from group receiving same pretreatment. * Significantly different (P < 0.05) from group receiving same exposure. 71 0SW 026465 STLCOPCB4010426 to TABLE n EFFECT OF AROCLOR 1254 AND CC14 ON SERUM TRANSAMINASES AND MICROSOMAL CYTOCHROME c REDUCTASE AND CYTOCHROME P-450 Treatment Na Cytochrome c Cytochrome SGPr1 reductaseb P-450c SGOT^ Corn oil--air Com oil--CCI4* Aroclor 1254*--air Aroclor 1254--CCI4 5 5 5 76 4.9 7S t 8.1 144 H.41 132 7.21 161 13.3 95 18.7h 522 37.3' 63 20.2h 13 2.3* 338 43.1*-h U 1.4 2213 193.4*-hJ 40 r,.f* 446 140.2b 33 * 2.3 2316 297.7h-1 a Number of rats. b nmoles of cytochrome c reduced/mg protein/min. c Difference in absorbance between 450 and 500 nm/mg protein X 10*. d Reitman-Frankel units * 4200 ppm for 2 h. f 25 mg/kg '.p. for 6 days. * 4 animals in this group. h Significantly different (P < 0.05) from group receiving same pretreatment. * Significantly different (P < 0.05) from group receiving same exposure. tCo O' *- O' O' STLCOPCB4010427 controls and in the Aroclor-pretreated groups although the magnitude of the change in the latter case (88%) was much greater than that seen in the controls (41%). Serum enzyme measurements were utilized as additional indicators of increased CCI4 toxicity. As indicated in Table 11, CC14 exposure increased SGPT and SCOT 26-fold and 11-fold, respectively. However, in the Aroclorpretreated rats these transaminases were elevated 200-fold and 70-fold, re spectively. Aroelor 1264 itself was without effect. To test the influence of the chlorine content on hepatotoxicity, Aroelor 1264 was compared with Aroelor 1221 and Aroelor 12C0 in separate experi ments. Two other serum enzymes, sorbitol dehydrogenase and isocitrate dehydrogenase were used in hopes of increasing the sensitivity. Preliminary experiments indicated no alteration of thei;e enzymes due to Aroelor 1254 alone. Therefore, Aroclor-pretreated rats exposed to air were not included in these studies. As indicated in Table III, exposure to 2900 ppm of CC14 for 2 h did not result in a significant change in glucose-6-phosphatase activity in the corn oil-pretrealed rats. Activity was decreased by 35% in the Aroelor 1221-prctreatcd animals. In tiro animals preheated with Aroelor 1254 the decrease in the enzyme activity due to CC14 was 70%. In the case of the sorbitol dehydrogenase, no activity was seen in the air controls, but, mea- TABLE III COMPARISON OF EFFECTS OF AROCLORS 1221, 1254 AND 1260 ON CC14 HEPATOTOXICITY . Treatment N* Corn oil--air Corn oil--CCI^ ' 1221,-CC14C 1 264r--CCU' ft 5 5 6 Glucosc-6 pliosphatascb 15.9 i 0.75 13.5 0.90 10.4 0.781-' 4.7 + o.'iO'-tk Sorbitol dehydrogenase0 0 55 22.3l 62 10.7* 495 + Bft.fthJ.k iRocitrate dehydrogenase1* 0 9 + 0.7h-1 15 1 1.6'-1 1324 i 4G5.11J'k Corn oil--air Corn oil--CC14 ]254,-CC)4* 12G0'-C14` 6 5 5 6 19.4 0.91 16.0 i 0.831 7.1 0.99`-l 6.7 1 O.OO1-' 0 40 1.11 309 + 36.3iJ 424 i 66.71-1 0 13 3 3.0` 670 -i 220.3lli 1110 349.5,J * Number of rats. b ^ moles POWfi/min. <.* IU/Io.ov um 103/0.2 ml scrum. * 2900 ppm for 2 h. * 26 mg/kg for 6 days. * 2900 ppm for 2 h. b 4 rats in this group. 1 Significantly different (P < 0.05) from corn oil--air group. I Significantly different (P < 0.05) from corn oil--CCI4 group. k Significantly different (P < 0.05) from other Aroelor prelreated group. 73 DSW 026467 . STLCOPCB4010428 surcable levels were found alter CCl 4 inhalation. These were not significantly altered by Aroclor 1221 pretrealment but were elevated 9-fold by Aroclor 1261 pretroatinent. Similar findings were seen with the isocitrate dehydro genase. Only slight activity was seen with CCl4 exposure in the corn oil controls. This was increased to a small degree (G6%) in the Aroclor 1221 pretreated rats but much elevated in the Aroclor 1254-pretreated animals (147-fold). Aroclor 1260 pretreatment was very similar to Aroclor 1254 (Table III). In none of the three parameters was there a statistical difference between the two Aroclors although they both showed remarkable potentiation of the changes due to CCl 4, Because of the impressive potentiating ability of Aroclor 1254 of the hepatotoxicity of CCl 4, it was of interest to determine if even lower doses of the Aroclor would enhance CCl 4 toxicity. Therefore, groups of rats were injected with 5, 10 or 25 mg/kg of Aroclor i.p. for 6 days. The results as presented in Table IV indicate thHt even at the dose of 5 mg/kg Aroclor 1254 potentiated the toxicity of CCl4 measured in terms of alterations in liver glucose-6-phosphalase and the serum enzymes sorbitol dehydrogenase and isocitrate dehydrogenase although in the case of the latter enzyme, the large standard errors prevented the nearly 8-fold potentiation from being statistically significant. It should also be noted that the potentiating ability appeared to be dose-related. Of further interest in view of the ability of Aroclor 1254 to potentiate CCl4 hepatotoxicity was determining if it would also potentiate the toxicity TABLE IV DOSE RESPONSE OF AROCLOR 1254 ON HEPATOTOXICITY Treatment N* Glucose-*)phosphatascb Sorbitol Isocitrale dehydrogenase' debydrogenaaed Corn oil-air Corn oil-CCI*' Aroclor 1264'--CCI4 5 mg/kg 10 mg/kg 26 mg/kg 8 18.6+0.61 8 13.2 + 0.47* 1 0.7 46 ? 9.6* 0 12+ 3.1* 8 9.6 + 0.51*-1, 132 + 27.0*'h 911 37.1* 8 8.3 0.41'-h 246 1 53.7*'h 819140.0,-h 8 5.8 i 0.16` h'i-` 340 i 40.8*'1''1 488 119.7th-1 Number of rots, b tiniolcs PC^/fj/inin. e 10s/0.2 ml scrum. III/). * 4 200 ppm for 2 h. I i.p. daily for 0 days. * Significantly different (P < 0.06) from corn oil -air. II Significantly different (P < 0.05) from corn oil- CCI4. 1 Significantly different (P < 0.05) from Arorlor 1 254--5 mg/kg ' Significantly different (P < 0.05) from Aroclor 1254 -- 10 mg/ii 74 DSW 026468 STLCOPCB4010429 TABLE V INFLUENCE OF CONCENTRATION OF CCI* ON POTENTIATION OF HEPATOTOXJCITY BY AROCLOH I 254 Pretrcotmcnl CCI 4" Nb (ppm) Olucosr-6* phosphatase' Sorbitol IsocitraU* dehydrogcnasert dehydrogenase1 Corn oil Corn oil Aroclor 1254 * Corn oil Aroclor 1264f 0 590 590 2350 2350 3 3 3 3 3 10.7 1.00 17.6 1,0.35 10.4 0.24*-h 16.1 + 0.52K 7.1 1 1.56*-'1 0 0 31 3.8'h 15 1 6.0'-' 148 i 16.0*,h'1 1 + 0.7 2 0.5 42 + ll.l*'h 8 1 7.3 288 t 113.6* * 2 h exposure. Number of rats. 0 /imolos PC^/g/min. <> A E^6"cm X 103/0.2 ml serum. * IU/l. 1 25 mg/Vg for 6 days. * Signifienntly different (P < 0.05) from corn oil--air. ** Signifienntly different (P < 0.05) from group with same exposure. 1 Significantly different (/' < 0,05) from group with same presentment. of a lower level of CCI 4 exposure. To determine this, animals were exposed to 690 ppm CCI 4 for 2 h and compared with animals exposed to 2350 ppm. The data in Table V indicate that at this lower level of exposure, no toxicity due to CCI4 alone was evidenced by any of the three parameters measured. However, in all three cases there were significant changes in the case of the Aroclor pretreated animals exposed to CCI4. As expected, there were changes due to CCI4 alone when it was inhaled at the higher concentration and these were further exacerbated in those animals pretreated with Aroclor 1254. DISCUSSION Although Bruckner ct ah (19] reported that Aroclor 1242 administration resulted in liver toxicity as evidenced by dovated SGOT values, the present experiments indicated little if any hepatotoxicity due to the Aroclor alone. This discrepancy is probably due to live fact that Bruckner ct al. used larger doses and/or longer time periods. CCI4 alone produced toxicity similar to other studies concerned with its inhalation f 14 ]. That Aroclor 1264 potentiates the toxicity of CCJ4 was demonstrated in all of the studies reported bore \s expected (ID] the administration of the Aroclor increased the liver to body weight ratio. This was further increased following CCI4 administration. Liver glucose-G-phosphatfise activity was de creased. The rises in the four serum enzymes that were measured were all greatly increased by the presentment with Aroclor 1254. The effects of Aroclor 1254 on dreg metabolism were us expected. 75 DSW Q26A69 Aroc.lor-induced increases in cytochrome r reductase and cytochrome P-450 have been shown by otiier workers 119] as lias the increase in /.-nitroanisole demothyinlion [9]. As with phcnobarhital | 4 ], neither the normal level of cytochrome c reductase nor flic elevated level following induction was al tered by CC1 ^ inhalation. However, similar to the case with phenoborbilal, there was a dramatic decrease in P-450 content, following CC14 exposure in those animals pretreated with the Aroclor 1254 as compared with the con trols. Similar comparisons ran be made with the effect on drug metabolism as evidenced by the large decrease in /i-nitroanisole demothylation. The comparison of the Aroclors was important in view of the findings of Chen and DuBois [9] that the ability of Aroclors 1221, 1254 and 1260 to induce A'-demelhylation and also EPN detoxification increased in proportion to the chlorine content of the Aroclor but that the O-demethylation of p-nitroanisole was induced to a greater extent by Aroclor 1254 than by Aroclor 1260. The resulk (Table 111) indicate that the two more chlorinated mixtures increase the hepatotoxicity of CC14 more than the less chlorinated Aroclor 1221, although no difference was delected between the two more chlorinated ones. An indication that Aroclor 1254 is a potent potentiator of CCI4 toxicity was indicated by the finding that a dose of only 5 mg/kg for 6 days was enough to demonstrate potentiation. ftecause of the difficulty in showing statistical significance clue to the large variation in response from animal to animal following CC14 inhalation, it did not seem worthwhile to give lower doses of the Aroclor. Anfher indication of its potency was the observation that even at a lower level of CC14 (590 ppm) than is usually employed in these studies there were significant elevations of the serum enzymes and decrease in liver glucose-6-phosphatase in the induced animals. A general conclusion that can be drawn from the results of these experi ments is that the Aroclors which induce P-448 rather than P-450 [7] arc very potent enhancers of CC14 hepatotoxicity so that care must be taken in considering the suggestion that 3-methykhoIanlhjene protects against CC14 by virtue of the fact that it induces P-448 [20]. ACKNOWLEDGEMENTS The author wishes to acknowledge the able technical assistance of Mrs. Barbara Schultz and Mrs. Nadylis Wood. The Aroclors were n gift of the Monsanto Company, St. Louis, Missouri. The work was supported by NIEHS Grant No. 00596. REFERENCES 1 A.E.M. McLean and E.K. 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