Document p2rMEOq3Rpvz8qXGmdOKrjg1j

Rough Draft Translation ABSORPTION AND DISTRIBUTION OF POLYCHLORINATED BIPHENYLS (PCB) AFTER INHALATORY APPLICATION* By H. F. Bcnthc, J. Knop, and A* Schmoldt Arch. Toxikol. 05-95 0972) Translated by Michael Dub, March 8, 1975 ABSTRACT ' ' In mala rats, after a single exposure to aerosol of a PCBmixture of low chlorinated biphenyls (Pydraul A 200, Monsanto), absorption and distribution were studied by measurements of PCB concentration in different organs. By this method of Inhaletory application a very good absorption is shown. Depending on cf TCB exposure vc observed c rapid increase of PCS concentration In liver; the concentration after 15 min. was already more than 50% of maximum concentration attained after nearly 2 hours (70 ug/g tissue). Concentration in fat after 50 min. of exposure is lk ug/g tissue (about 27% of liver con centration) whereas in brain tissue we found only 9 ug PCB/g tissue ( 17% of liver concentration) at this time. Dependent on time passed after the end of aerosal application we measured a rapid increase of PCB concentration In liver during 2k hours, accompanied by an Increase In levels In brain and fat. * The support of the project by the Federal Ministry for Youth, Family, and Health is acknowledged. - HONS 067453 1 In the course of 2 days brain and liver concentration fall to a minimum value, whereas In fat a maximum Is reached (200 ug/g tissue), which remains constant afterward. Thcro was no toxic fatty degeneration of liver under these experimental conditions* Explanation of PCB accumulation in liver and distribution from organ to fat is discussed. *' Polychlorinated biphenyls (PCB) have found a vide conmerclal application as plasti cisers in the production of plastics and lacquers, as a cooling agent and insulation material in electrical engineering, and as nonflaninable, anhydrous hydraulic fluids in mechanical conveying, for example, in mining. Depending on the physical requirements, mixtures with a chlorine content of 50-65% (dl-bis-heptachloroblphcnyl) have been used. A few Investigations of the toxicological properties of these substances have been available so far. Drinker et al. (1957) found that addition of the PCB vapors to the air in chronical tests with rats caused progressive liver damage, which Treon et el. (1956) were able to confirm after oral and Miller (19M) after percutaneous absorption in guinea pigs, rats, and rabbits. Chlorine-acne-llke contact dermati tis was observed in people (Meigs et al., 195*0. Further toxicological investiga tions, in view of the vide spread application, appear to be particularly required because, since 1966 in the residue analyses, besides pesticides, PCB are also found. They are found in the coastal water of North America (Riscbrough et al., 1968; Duke et al., 1970) and of Northern Europe (Holmes et al., 1967, Holden and Marsden, 1967; Kocman et al., 1969) in water, ocean floor, shells, algae, fish, sea mammals, and flsh-fcd birds and their eggs. -2 - HONS 067454 PCS** linvc also been detected In adipose tissue end in the mother's milk of humans; the concentrations arc In the same order of magnitude as that of DDT (Biros et al., 1970; Acker and Schulte, 1970). '' * In the commercial application of PCb'o as nonflammable hydraulic liquids, the inhala tion of the liquids presents the most frequent exposure form for man. During tech nical disorders (blow-out of Guperhcatcd couplings, leakages in pressure systems) very stable aerosols of these compounds are formed. In this manner extremely high concentrations in closed spaces may occur for relatively long periods of time. Since most toxicological investigations were carried out to date after oral, lntra- pcrltoncal, and cutaneous exposures to PCB, it appeared necessary to clarify the toxicological properties after an aerosol application in the animal tests. For these tests ve used the PCB mixture Pydraui A 200*1 (In the following referred to aa A 200) which la very frequently used aa a hydraulic fluid in mechanical con veying. Its cniorlne content amounts to **2e. Materials and Methods The teats were carried out on male Wistar rats (220-300 g), which 15-20 hours before the tests were deprived of food (Altromin). The aerosol was produced In an aerosol generator (Drager; particle size 0.5-JU at 3-5 atm) at 150*C. After passing through an empty wash bottle for cooling and separation of larger particles, the aerosol was conveyed through a short hose (20 ran diameter) into a closed rectangular plexi glass chamber 0*3 x 81 x 37 cm). The Inlet and outlet connections (20 ran diameter) were located near the floor In the opposite narrow sides of the chamber. As an animal container for a maximum 6 rets we used a vide-meshed lattice bar cage (plexiglass; 21 x 35 x 22 cm) which was placed in the special center of the aerosol ohamboT described above. 1 We acknowledge the generous donation of the substance by Monsanto Co. -3 - MCNS 067^55 For the entire duration of the teat the generator supplied a very stable uniform aerosol, and the air throughput amounted to 1 m3/hr. To determine the aerosol concentration, a preheated cylinder (1.5 1) was placed in the chamber, which after about 50 min. was closed and immediately was cooled In an ice-salt mixture. The precipitated PCB was dissolved in heptane, and the PCB concentration was determined photometrically in an aliquot part. The PCB determination carried out at different Intervals of the experiment gave a uniform value of 30.U g/m3. After the exposure to the aerosol, the animals'we^e killed either lamedlately or after a given tlma by a blow in the neck and decapitation. To that moment the animals obtained food end water in the desired quantities. The extraction of the tissue llpldes was carried out either immediately after removing the organs or after storing the OTgans at -25*C. The liver, before its removal, was rinsed with 0.9* NaCl for a complete blood removal from the abdominal aorta. For the analysis of the adipose tissue only the retroperitoneal adipose tissue wgs used. Since the llpldes are not further separated, the following date for fat and llplde ore always based on the total chloroform extract. A 1.0 g tissue sample was pulverized with anhydrous NasSOa to a dry powder and was extracted twice with 50 ml CHC13. The filtered CHC13 extracts were combined and were -concentrated to dryneaa in a rotary evaporator. The fat determination was carried out gravimctrlcally after drying over CaClg until a constant weight was obtained. About 50 *"8 the lipldc was weighed exactly and was boiled with 10% cthanolic potassium hydroxide for 2 hours under reflux end then was evaporated to dryness. The hydrolyzed fat was taken in 5 water, and the aqueous phase was oxtractcd with 6 ml heptane. Each time 5 heptane was removed, collected, and distilled to dryness in a rotary evaporator. The realdue was dissolved in 1.0 ml heptane. -h - MONS 067456 Since gns-Chromntographic measurement methods were not available, the heptane solution was fractionated by thin layer chromatography (silica gel with fluorescein indicator Riedel de Keen; eluent: twice with petroleum spirit, b.p. **0-60*C), the spots corresponding to the reference substance were extracted with a total of 5 ml heptane and were measured at 2^8 run in Spectrophotometer PMQ 2 (Zeiss). The UV spectrum of the PCB mixture used and those of both spots isolated from the chromatogram show an absorption band between 2^5 and 250 nm, which is suitable for a quantitative determination. The sum of the individually isolated chromatogram spots gave the same value as that of the same quantity of the known chromatographed starting substance. The sensitivity of the method was entirely sufficient for the investigations; the lower detection limit Is 0.3 Ug/ml. Moreover, the tin layer chromatography has an advantage because further expensive "cleanup" methods are not required anymore. The yield of the A 200 quantity added to the tissue homogenates (15-75 ug/g tissue) amounted to: 72.6 + 6.6% for liver, 75*1 5.1% for brain, and 69.I + 8.0% for adipose tissue. For the calculations, a uniform value of 72% was Ucu. ( To determine the glycogen contente of the liver, kOO mg tissue wes boiled with 3 ml N KOH for 20 min. and then was cantrifuged. The sediment was suspended in 3 ml 96% ethanol and was reprecipitated at the boiling temperature. The glycogen was hydrolyzed with 2 ml U N HC1 at 100*C during 60 minutes, the hydrolysis product was neutralized with KOH and and was filled to 100 ml. The determination of glucose thus obtained from glycogen was carried out ensymically with hexoklnasc, glucosc-C-phosphatedchydrogcnase,,and NAD?, by measuring the resulting NADPH at 366 nm. All the chemicals were of the analytical grade, the organic solvents were spectrally pure (Uvasol Merck), the glucose determination was carried out by the glucose test combination (Bochringcr Mannheim). -5- HONS 06 7*5? The statistical evaluation of the tests on k-6 animals was carried out according to the uncomblncd t-tcat. An error probability of 5% is assumed. , * RESULTS Since FCB s are considered as toxic to liver, the question of the absorption of Tydraul A TOO in the liver was the central point of the investigation. As Figure 1 shows, an exposure time of 15 min. to aerosol was sufficient for achiev ing more than 50% of the maximum deposition/^he liver. After that the absorption curve has a considerably flatter course and after 2 hours approaches the value of 69 7 ug/g liver wet weight, the maximum concentration. Knowing this rapid absorption by the liver, the exposure time In the following investigations wss limited to 5O minutes. In the investigation of the questionhow long the applied PCB mixture remains stored unchanged in the liver, the test animals were killed not lnmcdistely after the exposure to the aerosol, but after 12 and 2k hours, respectively. During that time the rats were fed again. Since feeding after 2k hours fasting (before the exposure to the aorosol) leads to physiological changes of the liver weight and of the fat contents, the data of the A 200 concentration were given in ug/g liver llpidc. Figure 1. Absorption of A 200 in the liver as a function of the exposure time. The average value of 6 rots + sx. -6- H0KS 067458 figure 2 chows Che concentration decrease os a function of time. After 12 hours the contents decreased already to less than a half of the starting quantity. After 2 days the PCB concentration fell to a value of 9^ liver lipldc. These findings left open the question whether this involves a metabolic process, excretion, or a redistribution into the other organs. Since PCB's ore highly lipophilic substances which are found, besides pesticides, in the animal adipose tissue, the retroperitoneal adipose tissue of rat was analyzed under the same experimental conditions aa in the liver tests. It was established thereby that Immediately after the aerosol exposure with I9.3 ug/g llplda (corresponding to 1** ug/g adipose tissue) only traces of PCB were detected in the adipose tissue. After 12 hours only a slight concentration Increase is measurable, and only after 36 hours the A 200 contents reaches s maximum of 352 ug/g fat. After 72 hours this value is not essentially lower (Figure 3). Thcso findings speak against the only elimination by the liver that has not been clear, rather it appoars that first a transfer Into the adipose tissue takes place. Figure 2. The A 200 concentration decrease of the liver after 30 minutes aerosol exposure as a function of time. The average values for 6 rats + sx. -7 - MCNS 067459 As tn the case of DDT, it Is here also of particular toxicological interest to find out whether PCB's can be detected in the brain. Again the same experimental conditions were chosen. In contrast to the findings in the adipose tissue, Immediately after the exposure to A 200, the latter could be detected in the brain. The measurements as a function of time after the exposure (Figure k) gave a concen tration increase reaching a value of 226 ug/g brain lipldc after 2k hours. After that there occurred a decrease dovn to the concentration equilibrium with the liver lipldc. .. \ To verify whether A 200 in the form of the inhaled aerosol leads to a toxic fatty degeneration of liver, the liver fat contents was determined as e function of the exposure time and was compared with the control animals . It was necessary thereby to observo that the'control animals also remained fasting during 2k hours, since the fat contents Increased during fasting (Ekmann, 19^9). It was verified by means of tha liver glycogon contents that here a dienimllating metabolism condition was Involved, since after 2k hours of fasting the glycogen contents decreased to zero (in normally fed control animals J.5 0.9 g %) As another conditionally valid parameter of a liver degeneration, the relative liver weight was measured. Table 1 shows that even an aerosol exposure of 2 hours does not lead to any significant change either In the liver fet contents or in the relative weight of the liver,as compared with control animals. -8- AOfcS 06 7460 Figure The concentration course of A 200 In the adipose tissue after 30 min. aerosol exposure as a function of time. The average values of 6 rats + sx. + a significant difference of the adjoining values. Figuro k. The A 200 concentration in the brain as a function of time after 30 min. aerosol exposure. The average values of 5 tats + sx. + a significant difference of adjoining values. -9 - *CHS 6i Table 1. The behavior of the liver adipose contcntn (g %) and of the rdntlvo liver weight (r, % body weight) ns n function of the exposure duration to A 200 aerosol. The average values of 6 rate a. Control Normally Fed Control 2k Hr. F.-lStlW! Duration of the Aerosol Exposure 15 Min. 30 Min. 60 Min. 120 Min. Liver Fat (b *> Relative Liver wt. (a 3.6 + O.k 3.8 + 0.3 k.9 + 0.8 2.8 + 0.1 k.7 0.6 3.1 +0.5 k.8 + 0.5 2.9 + 0.2 k.3 + 0.5 5.0 + o.i 5-1 + 0.1 3.0 + 0.2 Table 2. The behavior of the liver adipose contents (c%) and of the relative liver vcir.ht (g Z body weight) after 30 min . aerosol exposure as a function of time. The averace values of 6 rats ~ s* Liver Fat (c *> Relative Liver Wt. (b V Control . 2k Hrs. Fastlns, Control 2k Hrs. Feeding 2k Hre. Fasting 3.0 + 0..2 k.9 + 0.8 k.5 + O.k 2.8 + 0.1 Tima After 30 Min. Exposure to Aerosol 0 Hr. 12 Hrs. 2k Hrs. Hrs. 78 Hrs. k.8 +0.5 3.k + O.k 2.9 + 0.3 2.8 + O.k 3.0 + 0. 2.9 0.2 5.0 + 0.3 k.2 + 0.5 k.O + O.k k.3 + o. To verify whether the liver changes occur perhaps after a certain delay, the parameters were followed up to 72 hours after an exposure of 30 minutes. The values given In Table 2 do not differ from the control data. The decrease of the llplde values to the subnormal values after the renewed feeding can bo explained as a build-up process. - 10 - MGNS 06746 DISCUSSION During suddenly occurring leakages In the hydraulic pressure system (Approximately 120 atm.) and in heat exchangers maintained under pressure there are formed very stable aerosols to vhich the operating perconnel may be exposed for a longer time. To imitate such situations in model experiments, rats were exposed only once for s relatively short time to the FCB aerosol. In addition, these tests had the advantage of allowing us to study the kinetics of the TCB distribution. The rapid accumulation in the liver and the measurement of the maximum concentration *\ immediately after the exposure Indicate that PCB enters Immediately Into the blood circulation and Is not deposited first in the alveoli (see Figures 1 and 2). Bccausc of the lipophilic properties and the structural relation to DDT, above all the distribution was investigated In those organs which are also of main toxicological Interest for DDT and similar pesticides. These organs include brain, liver, and adipose tissue (Kagan, 1969). In the previous investigations there was reported particularly about the liver toxicity of PCB's (Drinker et al., 1937; Treon et al., 1956; Killer, 19^)* The investigations which were primarily pathohistologleal left out, however, the quantitative PCB analyses of the organs. While during the enteric resorption a high PCB concentration In the liver Is to be expected, it was surprising to also find the highest concentration in the liver during the aerosol exposure. In contrast, in spite of the many times higher blood circulation, we found in the brain only 11% of the liver concentration (based on ppm lipldes) and 17% (based on ppm wet tissue). Inmcdlately after exposure, only traces of PCB are detectable in the adipose tissue. During the time following the exposure, the distribution pattern changed. Within the first 2** hours the PCB content in the liver decreases exponentially, while at the same time the concen tration in the brain and in the adipose tissue increases to the same value (based on the ppm lipidc). In the brain, however, the contents decreases after ^8 hours below the starting value and reaches,M^the liver, a level of 80 ppm lipidc, which - 11 - 067463 i". bcth crgr.r.s dcca not change significantly anymore, even after 72 hour*. In the adipose tissue the maximum is reached only on the second day after the exposure. The value of 352 ppm does not significantly decrease 6n the third day findings can be explained only aa a redistribution of PCB from the liver into the adipose tissue. . It Is not clear why the applied PCB mixture initially accumulates predominantly in the liver. The higher blood circulation through the liver elone cannot explain the accumulation. The brain which has even betted blood supply reaches, during the 30 minutes lasting . exposure, only 17^ of the concentration that la measured in the liver tissue. The brain concentration Increases, however, in the phase of the redistribution from the liver into the adipose tissue, although the plasma concentration is then lower than at the time of exposure. It Is possible that for the explanation of the PCB enrichment In the liver one may consider the condition that the rats were fasting for 20 hours prior to the test. Perhaps this pciiou of time is sufficient lot adjusting the metabolism to the conditions of fasting. ?or the fat metabolism this means an increase of llpolysls with simul taneously inhibited absorption of triglycerides In the adipose tissue. The nonesterlfled fatty acids set free combine with plasma albumen and are absorbed by. the liver whore they serve a a the energy source and, due to an inhibited lipoprotein synthesis,arc deposited in Increased quantity as triglycerides (Tarnowski and Seitz, 1970). Parallel to the Increased triglyceride concentration of the liver, an increased PCB absorption may occur. Preliminary results of the Investigations with fed rats, under otherwise the same experimental conditions, confirm this hypothesis so far as the PCB concentration of the liver at the beginning was actually found somewhat lower, nevertheless It is still higher than in the other organs. Nothing has yet been published on the PCB redistribution from the liver into the adipose tissue. Crant at al. (1971) reported about the distribution after oral 12 - HONS 067464 Application and found, In the case of this longer lasting absorption In the adipose tissue lnmcdlatcly from the beginning of the experiment, higher values than those of the liver. '* ` It is also not known to what extent, besides the redistribution, the elimination and metabolic proccssco take place. According to the investigations of the enteric resorption in the presence of the esthetes Introduced Into the bile duct and into the bladder, within the first 9 hours no excretion of unchanged PCB could be detected (Benthe and Schmoldt, in preparation).' ft is known about afho elimination of PCB's after a prolonged period of time that the compounds with a lower chlorine content ore moro rapidly eliminated than are the higher chlorinated biphenyls (Grant ct *1., 1970- The liver toxicity cited above la manifested in a central atrophy and an adipose degeneration as well as in a porphyry (deVos and Kocman, 1970). Under the expert* iiiumLuI cw.iulLio.iS of a single, chert-lasting seroscl exposure there not fovnd, however, either a decrease of the liplde contents or an increase in the relative liver weight. Likewise, no pathological changes were detectable histologically by light microscopy. However, other investigations (Nissan, 1971) Indicate that during longer exposure tints the fat contents of the liver Increases and the liver functions Arc pathologically changed. - 13 - HONS 067465 REFERENCES Acker, I.., Schulic, E.! tl'ff das \ orkommen von chloricrtcn D'phrnylcn und lien* chiorhctuol nehen chlnrirrlcn ln.*rktir.iilrn in Ihimnnnnlch und mrmchiithcm Peltpcwrbc. N.itur iirm{hnilrn .77. 407 (IOTOi. Drnthe, II. F., Shmoldt, A.: Rennie und biliarc Elimination von polychloricrtcn lliphrnylen lI'CH). In Vorbercitung. Biros, F. J., W .ilkcr, A. D., Medbery, A.t Polychlorinated bipbcnyl* in human dipofc litviip. Hull. Environ. Conltn. Toxicol, 4, 317 (1970). Drinker, C. K.. Warren. M. F., Rennet, 0. A.: The problem of powihlc lytlemic effect* from erruin rblorinotcd hydrocarbons. J. industr. Jlye. 19, 233 (1037). 1 Duke, T. W., lytue, J. I., W ilton, A. J., Jr.: A i>olvcMr>niwitcd biphenyl (Aroelor 12.74*) in the water, sediment, ami biota of Escambia Content. Toxirof. 5, 171 (1970). Ekmann, C. A.. Holmgren, 11.t The effect of alimentary factors on rlyeogrnrhythma and distribution of glycogen in the liver lobule. Anat. Rrc. 101, 160 (1049). Ctant, D. L-, Phillip*. \V. K. J., Villeneuee, D. C.: Metabolism of a polychlorinated ,biplirnvl (Aroelor 12&4) mixture in the rat. Bull. Environ. Contain. Toxieol. 0 103 (1071). Holden, A. V.. Marsdrn, K.: Organocblorino poalicidea in aeale tod porpoise*. Mature (loml.) SIR. 1374 (1007). lloltner, D. C.. .Simmon*, J. 11., Tation, J. O. C.t Chlorinated hydrocarbon* in Uritiah wildlife. Nature (Lorn) ) SIC. 227 (1007). Kagan, V. S.r Harmful effect of DDT. Retiduc Rev. 27, 36 (1000). Koeraan, J. If., Ten NocTrr de Braun-.M. 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J. 17, 204 (1060). Voe, G. H. Kocmann, J. H.t Comparative toxicologic study with polychlorinated .JAphcuyl* in chickens with special reference to porphyria, edema formation, liver nacrosis, and tiaawo residues. Toxicol, appl. Pharmacol. 17, 660 (1070). Prof. Dr. H. F. Ilcnthe Pliarmakologisches lnatitut ' dec Universitht , Uoircrsitnts-Krnnkcnhaua Eppendorf D-2000 Hamburg 20 MartlnialraOe 02 .DeuUakland - lU - HONS 067466