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The effect of food restriction on the redistribution of hexachlorobenzene in the rat
TOXICOLOGY
AND
The
APPLIED
PHARMACOLOGY
Effect
31, 3 13-319
(1975)
of Food Restriction of Hexachlorobenzene
on the Redistribution in the Rat
DAVID C. VILLENEUVE Bureau
of Chemical Safety, Health Ottawa, Ontario, Canada
Received
July
10, 1974,
accepted
Protection KIA OL2 Jrrly
Branch,
29, 1974
The Effect of Food Restriction on the Redistribution of Hexachlorobenzene in the Rat. VILLENEUVE, D. C. (1975). Toxicol. Appl. Pharmacol. 31? 313-319.Adult malerats were given hexachlorobenzene(HCB) daily (PO) at dosesof 0, 1.0, 10,and 100mg/kg for 14 days.The diet of theseanimals wasthen restricted to 25% of their normal food intake for a period of 10 daysafter which all animalswerekilled and their tissuesremovedfor HCB analysis.Food restriction causeda mobilization of the HCB storedwithin the fat depot which resultedin a transfer of this compoundinto the plasma and other tissuesof the body. Toxic signsincludinglossof appetite,tremors, and death occurred in the group of animalsreceiving 100mg HCBjkg and subjectedto food restriction. The tissueresidueprofile indicatedthat death occurred when brain concentralion exceeded300ppm. Residuesof many pesticidesincluding hexachlorobenzene (HCB) occur in human and animal adiposetissueaswell asin the lipid material of other organs(Mrak Report, 1969; Acker and Schulte, 1970; Koeman et al., 1969; Brady and Siyali, 1972; Siyali, 1972). Since the adiposetissuetends to act as a reservoir for thesecompounds, depletion of fat depots can result in the mobilization and redistribution of the stored pesticide. In studies carried out by Dale et al. (1963) the lo-day starvation of rats previously fed 200 ppm DDT resulted in elevated levels of residue in the brain. Similarly, Ecobichon and Saschenbrecker(1969) demonstrated that food deprivation in cockerels causedthe mobilization of DDT from the fat depot to other tissuesincluding the brain and resulted in tremors, convulsions and death. It is obvious from theseand other studies(Zabik and Schemmel, 1973; Clausen and Konat, 1972) that weight loss from whatever the cause can result in a dramatic redistribution of the pesticidescontained in the adipose tissue and if the initial level of thesepesticidesis sufficiently high, toxic signsmay occur. Since many people restrict their diets either voluntarily or becauseof illness, then tissue redistribution of pesticide burdens assumesa potential health hazard. In a preliminary study carried out in this laboratory five out of six rats which had been dosedwith HCB (100 mg/kg daily for 14 days) died 3-9 days after having their food intake restricted to 2576. The present study, therefore, was initiated to (1) confirm the toxic effects observed in the preliminary study; (2) determine whether such effects occur when HCB is administered at lower levels; (3) measure the extent of HCB excretion during administration and food restriction; and (4) define the extent of tissueredistribution of HCB in animals subjected to food restriction. Copyright [I 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
313
314
DAVID
C. VILLENEUVE
METHODS Sixty male Sprague-Dawley rats (Bio Breeding Laboratories Inc., Ottawa, Ontario) weighing 375-420 g were divided randomly into four groups and given HCB (BDH Chemicals Ltd., Britain) po in corn oil (0.5 ml/100 g body wt) daily for 14 days at 0, 1.O, 10, and 100 mg/kg body wt. During this period the animals were fed ad lib. with standard laboratory feed (Master Feed). Food consumption was monitored and feces collected from each animal daily. Twenty-four hours after the last dose was administered five animals from each group were killed (Group 1) and the following tissues removed for HCB analysis; blood, liver, kidney, fat, heart, lung, spleen, brain, and muscle. Five animals from each group were placed on 25 “/, of their normal food intake for 9 days (Group 3) while the five remaining animals were allowed to feed ad lib. during the same period (Group 2). Urine and feces were collected daily from each animal and on the tenth day after dosing ceased all animals were killed and their tissues removed, weighed, and frozen pending HCB analysis. Tissues were analyzed for HCB content using a glc-EC procedure described earlier (Villeneuve et al., 1974) and results expressed as ppm HCB based on the wet tissue weight. All food consumption, body and tissue weight data were subjected to statistical analysis using Student’s t test. RESULTS None of the animals in the 0, 1.O, or 10 mg/kg HCB groups died as a result of dosing or food restriction. With those animals receiving 100 mg/kg HCB, none died during the 14-day dosing period. However, one animal of this group which was allowed to feed ad lib. subsequent to dosing died 4 days after receiving the last dose of HCB. Of the animals dosed with 100 mg HCBjkg and then placed on 25 % of their normal food intake, four died. Of these, one animal died 3 days after dosing ceased, two rats died 6 days after, and one died 10 days after. In all cases the animals stopped eating 1-2 days prior to the onset of death, and displayed tremors and a reddish nasal discharge. HCB treatment did not affect body weight gain over the 14-day dosing period. The animals which were dosed with HCB at 10 mg/kg and then fed ad lib. (Group 2) gained more weight over the 9-day period than their respective controls. Only animals which received 100 mg/kg showed an increased weight loss when they were placed on 25 % of their normal food intake. Food consumption during the dosing period was not affected by HCB administration. The tissue weights, expressed as percent body weight are shown in Table 1. Food restriction did not affect spleen, brain, kidney, or heart weights but significantly reduced liver weights (p < 0.05) when expressed as a percent of body weight. HCB treatment did not affect brain, kidney, or heart weights at any dose level when expressed as percent body weight. Spleen weight was increased in those animals receiving 100 mg/kg while liver weights were increased at both 10 and 100 mg/kg. In those animals subjected to both HCB and food restriction, increased brain weights (percent body weight) were observed at 10 and 100 mg/kg; increased kidney weights (percent body weight) were observed at 100 mg/kg. HCB administration at both 10 and 100 mg/kg negated the effect of lowered liver/body weights observed during food restriction.
FOOD
RESTRICTION
AND
HCB
1N THE
31.5
RAT
TABLE 1 TISSUE WEIGHTS (%
BODY WEIGHT) OF RATS TREATED HEXACHLOROBENZENE (HCB)
WITH
Heart
Liver
0.32 + 0.01 0.28 k 0.02 0.28 + 0.01
3.59 + 0.08 3.89 + 0.29 2.37 + 0.10
0.31 + 0.01 0.28 + 0.01 0.29 + 0.02
3.48 & 0.06 3.69 + 0.12 2.49 + 0.03
0.75 4 0.02 0.73 * 0.03 0.78 f 0.03
0.32 k 0.01 0.28 k 0.01 0.28 f 0.01
3.97 * o.12c 4.18 t 0.10 4.11 * 0.13’
100 mg/kg HCB 0.44 & 0.02 0.77 2 0.01 0.47 * 0.01 0.80 +_ 0.02 0.67 + 0.03’ 0.89 f 0.02’
0.28 + 0.09 0.28 f 0.02 0.31 + 0.01
5.85 + 0.14’ 6.83 + 0.28’ 6.74 + 0.48’
Groupb
Spleen
Brain
Kidney
1
0.16 + 0.01
2 3
0.15 + 0.01 0.14 + 0.01
0.46 + 0.02 0.45 + 0.03 0.52 + 0.03
1 2 3
0.14 2 0.01 0.14 * 0.01 0.14 * 0.01
0.40 + 0.01 0.55 * 0.02
1 2 3
0.18 f 0.03 0.14 -t 0.02 0.16 + 0.01
0.47 4 0.01 0.46 4 0.03 0.64 + 0.04”
1
0.19 + 0.03’ 0.19 t 0.01’ 0.20 + 0.07’
0 mg/kg HCB 0.72 + 0.02 0.67 f 0.02 0.71 + 0.01
1.Omg/kg HCB 0.50 + 0.01 0.78 + 0.03 0.74 + 0.02 0.73 + 0.02
10 mg/kg HCB
2 3
n Values represent mean t SE of 5 animals. b See text for details of treatment for groups 1, 2, and 3. c Denotes significant difference from 0 group at p i 0.05.
The tissue residue data obtained from the animals dosed 1, 10, and 100 mg/kg is shown in Table 2. In nearly all tissues the concentration of HCB was lower in animals fed ad lib. for 9 days (Group 2) than those killed immediately after dosing (Group 1). Two exceptions to this were fat and muscle tissue. The concentration of HCB was higher in all tissues from animals whose food intake was restricted (Group 3) compared to those fed ad lib. (Group 2). The tissues showing the greatest proportional increase in HCB after food restriction were brain, spleen, liver, and heart followed by fat, plasma, kidney, muscle, and lung. When results were calculated in terms of total micrograms of HCB per tissue, brain again showed the greatest increase foIlowed by liver, spleen, heart, and kidney. All tissues examined accumulated HCB in a dose-dependent manner and levels of residue were always highest in adipose tissue followed by liver, kidney, lung, brain, spleen, heart, muscle, and plasma. The fecal excretion of HCB during the dosing and subsequent food restriction is shown in Fig. 1. The amount of HCB excretion in the first 24 hr for all three doses was approximately 1 “/0 of the amount administered. The excretion profiles were similar for all three levels during the dosing period. Although there was considerable variation in the amount of HCB excreted on a day to day basis, there was no obvious trend toward increased excretion throughout the dosing period. After the last dose of HCB was administered there was a sharp drop in the amount excreted. In the group of animals dosed 1 mg/kg, the animals subjected to food restriction excreted less than the animals fed ad lib. At the 10 and 100 mg/kg levels, the animals subjected to food restriction excreted HCB in amounts similar to those fed ad lib.
20.3 -t 1.4 17.4 * 1.4 191.5 k 25
117+3 83 + 11” 385 + 38
18.9 + 2 13.1 * 0.9 48.2 L- 6
109 + 11 62f6” 309 * 9
2.2 * 0.2 2.8 to.2 9.2 + 1.4
17.9 L- 0.8 12.9 + 1.5’ 43.ge
3.1 F0.4 4.4 +- 0.3 5.5 & 1.4
2.8 + 0.5 1.3 f 0.1 4.1 If: 0.8
0.43 & 0.07 0.31 + 0.01 0.71 to.15
Brain
Spleen
PROFILE
Plasma
RESIDUE
OF
Kidney
177* 11 129 k 11” 467 + 110
175 + 10 90 rt 3d 339 + 26
10mg 34.3 & 2.4 34.5 -t 3.4 46.4 + 5.3 30.2 + 3.5 68.2 _+ 6.3 68.2 _t 6.2 100 mg
-
.-
13.8 f 0.7 20.6 + 6 49.2 + 17 69 &- 6 84 + 18’ 282 f- 170
96 f 2 70 + 3d 297 k 23
2.7 + 0.4 1.2 1.1 2.5 + 0.3 +
Musde
(HCB)
17.2 + 1.4 15.1 * 1.1 66.6 f 12
2.4 k 0.3 1.5 0.1 4.2 f 0.7 +
Heart
HEXACHLOROBENZENE
1.0 mg 4.7 + 0.7 7.7 + 1.0 4.4 -f. 0.3 3.1 + 0.2 1.4 4.8 1.0 6.1 + +
Lung
WITH
HCB (ppm wet weight)
RATS TREATED
2
’ Mean residue concentration &SE of 5 animals except where otherwise indicated. b See text for details of treatment for groups 1, 2, and 3. ’ Mean &SE of 4 animals. d Mean &SE of 3 animals. e Value from I animal only.
Groupb
TISSUE
TABLE
268 t 15 167 + 22” 614 + 70
58.6 & 1.9 35.1 +- 2.0 176 rt 51
5.7 + 0.5 2.9 0.2 7.7 + f 1.4
Liver-
3860 -t 575 3869 + 360’ 10,597’
527 + 32 667 + 61 2204 & 426
87 + 14 59.4 * 4 161 + 6
Fat
is
C
: f u P -e F E;; 3
FOOD
RESTRICTION
AND
HCB
IN
THE
317
RAT
DOSING STOPPED
I1
5
I
I
I,
I,,
IO
,
DAYS
,
15
,
,
,
,
,
,
20
FIG. 1. Daily fecal excretion mg/animal of HCB during dosing and subsequent period of food restriction. (0-U) 100 mg HCBjkg body wt; (A-A) 10 mg HCB/kg body wt; (x-x) 1 .Omg HCB/kg body wt. The solid line (-) after dosingrepresents animals fed ad lib. The broken line (-- -) represent animals on l/4 normal food intake. Each value during dosing represents the mean daily excretion of 15 animals. Each value after dosing represents the mean daily excretion of 5 animals.
DISCUSSION Hexachlorobenzene has recently gained prominence as an environmental contaminant through its use as a fungicide and an industrial chemical. Like DDT, and other members of the organochlorine group, HCB demonstrates a high persistence,limited biodegradibility, and considerable lipophilicity. Becauseof its structural and chemical similarity to the DDT-type compounds the present study was carried out to determine the extent to which HCB is relocated when fat depots in animals previously exposed to HCB are mobilized. The results in this study confirmed the toxic effects observed in a preliminary study. Toxic signswere observed only in animals dosed 100mg/kg for 14consecutive days. No toxic signswere observed in animals dosed at the other levels except for an increased liver/body weight ratio at 10 mg/kg. Animals which died after receiving 100mg/kg body weight first displayed a lossof appetite, tremors, and a red nasaldischarge.This reddish discharge has been noted in acute DDT poisoning and was attributed to oversecretion
318
DAVID
C. VILLENEUVE
of porphyrin by the Harderian glands (Henderson and Woolley, 1970). The combination of HCB administration and food restriction increased spleen weight at 100 mg/kg and increased liver weight at 10 and 100 mg/kg. The latter observation can be related to the ability of HCB to cause liver hypertrophy (Grant et al., 1974). The residue profile indicates that HCB is accumulated in all tissuesexamined in a dose dependent manner. Toxic signsand death were observed in animals whose brain residue levels were in excessof 300 ppm. These results agree with data from the preliminary study where the five animals which died had brain HCB concentrations of approximately 400 ppm while the lone survivor had a brain level of 266 ppm. The one animal which died after being treated with HCB at 100 mg/kg followed by normal food intake had lost 180 g and had a brain concentration of 360 ppm HCB. Fecal analysis indicated that food restriction did not increase the excretion of HCB and thus total body burdens of HCB are not drastically reduced as has been reported for DDT and dieldrin (MacDonald ef al., 1973). Urinary excretion was not monitored in the present study becauseresults from the preliminary experiment indicated that most of the HCB was excreted in the fecesand that the urinary excretion pattern wasessentially the same as for the fecesduring HCB administration and the subsequentperiod of food restriction. The residuedata indicate the concentrations of HCB observed in the brain of dead animals (>300 ppm) are considerably higher than those observed for rats poisoned with DDT [35 ppm (Dale et al., 1962)].
ACKNOWLEDGMENTS The technical assistance of Miss Luz Panopio and Mr. Henry Jamesis gratefully acknowl-
edged. REFERENCES ACKER,
L. AND
SCHULTE,
E. (1970). Appearance of chlorinated biphenylsand hexachloro-
benzene along with insecticides in human milk and fat tissues. Nuturwissenschaften 57, 497. BRADY, M. N. AND SIYALI, D. S. (1972)Hexachlorobenzene in humanbody fat. Med. J. Aust. 1, 158-161. CLAUSEN,
J. AND
KONAT,
G. (1972) Enzymic and behavioral changesinduced in mice fed
polychlorinated biocides followed by starvation. Experientia 28, 902-903. DALE, W. E., GAINES, T. B. and HAYES, W. J. (1963). Storage and excretion of DDT in starved rats. Toxicol. Appl. Pharmacol. 4, 89-106. ECOBICHON, D. J. AND SASCHENBRECKER, P. W. (1969).The redistribution of stored DDT in cockerels under the influence of food deprivation. Toxicol. Appl. Phramacol. 15, 420-432. GRANT, D. L., IVERSON, F., HATINA, G. V. AND VILLENEUVE, D. C. (1974)Effects of hexachlorobenzene on liver porphyrin levels and microsomal enzymes in the rat. Environ. Physiol. Biothem. 4, 159-165. HENDERSON, G. L. AND WOOLLEY, D. E. (1970). Mechanisms of neurotoxic action of 1,1,1trichloro-2.2-bis(p-chlorophenyl) ethane (DDT) in immature and adult rats. J. Pharmacol. Exp. Ther. 175, 60-68. KOEMAN, J. H. TEN NOEUVER DEBRAUUS, AND DE Vos, R. H. (1969). Chlorinated biphenyls in fish mussels and birds from the river Rhine and the Netherlands coastal area. Nature (London) 221, 1126-l 128. MACDONALD, W. E., DEICHMANN, W. B. CUBIT, D. A. AND BEASLEY, A. G. (1973). In Pesticides and the Environment: A Continuing Controversy, pp. 87-101. Intercontinental Book Corporation, New York.
FOOD
RESTRICTION
AND
HCB
IN THE RAT
319
Report of the Secretary’s Commission on Pesticides and their Relationship to Environmental Health, U.S. Dept. of Health, Education and Welfare, December 1969 (Mrak Report). SIYALI, D. S. (1972). Hexachlorobenzene and other organochlorine pesticides in human blood. Med. J. Artst. 2, 1063-1066. VILLENEUVE, D. C., PHILLIPS, W. E. J., PANOPIO, L. G., MENDOZA, C. E., HATINA, G. V. AND GRANT, D. L. (1974). The effects of phenobarbital and carbon tetrachloride on the rate of decline of body burdens of hexachlorobenzene in the rat. Arch. Environ. Contamitt. Toxicol., 2,243-252. ZABIK, M. E. AND SCHEMMEL, R. (1973). Dieldrin storage of obese, normal and semistarved rats. Arch. Environ. Health 27, 25-30.
AND
The
APPLIED
PHARMACOLOGY
Effect
31, 3 13-319
(1975)
of Food Restriction of Hexachlorobenzene
on the Redistribution in the Rat
DAVID C. VILLENEUVE Bureau
of Chemical Safety, Health Ottawa, Ontario, Canada
Received
July
10, 1974,
accepted
Protection KIA OL2 Jrrly
Branch,
29, 1974
The Effect of Food Restriction on the Redistribution of Hexachlorobenzene in the Rat. VILLENEUVE, D. C. (1975). Toxicol. Appl. Pharmacol. 31? 313-319.Adult malerats were given hexachlorobenzene(HCB) daily (PO) at dosesof 0, 1.0, 10,and 100mg/kg for 14 days.The diet of theseanimals wasthen restricted to 25% of their normal food intake for a period of 10 daysafter which all animalswerekilled and their tissuesremovedfor HCB analysis.Food restriction causeda mobilization of the HCB storedwithin the fat depot which resultedin a transfer of this compoundinto the plasma and other tissuesof the body. Toxic signsincludinglossof appetite,tremors, and death occurred in the group of animalsreceiving 100mg HCBjkg and subjectedto food restriction. The tissueresidueprofile indicatedthat death occurred when brain concentralion exceeded300ppm. Residuesof many pesticidesincluding hexachlorobenzene (HCB) occur in human and animal adiposetissueaswell asin the lipid material of other organs(Mrak Report, 1969; Acker and Schulte, 1970; Koeman et al., 1969; Brady and Siyali, 1972; Siyali, 1972). Since the adiposetissuetends to act as a reservoir for thesecompounds, depletion of fat depots can result in the mobilization and redistribution of the stored pesticide. In studies carried out by Dale et al. (1963) the lo-day starvation of rats previously fed 200 ppm DDT resulted in elevated levels of residue in the brain. Similarly, Ecobichon and Saschenbrecker(1969) demonstrated that food deprivation in cockerels causedthe mobilization of DDT from the fat depot to other tissuesincluding the brain and resulted in tremors, convulsions and death. It is obvious from theseand other studies(Zabik and Schemmel, 1973; Clausen and Konat, 1972) that weight loss from whatever the cause can result in a dramatic redistribution of the pesticidescontained in the adipose tissue and if the initial level of thesepesticidesis sufficiently high, toxic signsmay occur. Since many people restrict their diets either voluntarily or becauseof illness, then tissue redistribution of pesticide burdens assumesa potential health hazard. In a preliminary study carried out in this laboratory five out of six rats which had been dosedwith HCB (100 mg/kg daily for 14 days) died 3-9 days after having their food intake restricted to 2576. The present study, therefore, was initiated to (1) confirm the toxic effects observed in the preliminary study; (2) determine whether such effects occur when HCB is administered at lower levels; (3) measure the extent of HCB excretion during administration and food restriction; and (4) define the extent of tissueredistribution of HCB in animals subjected to food restriction. Copyright [I 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
313
314
DAVID
C. VILLENEUVE
METHODS Sixty male Sprague-Dawley rats (Bio Breeding Laboratories Inc., Ottawa, Ontario) weighing 375-420 g were divided randomly into four groups and given HCB (BDH Chemicals Ltd., Britain) po in corn oil (0.5 ml/100 g body wt) daily for 14 days at 0, 1.O, 10, and 100 mg/kg body wt. During this period the animals were fed ad lib. with standard laboratory feed (Master Feed). Food consumption was monitored and feces collected from each animal daily. Twenty-four hours after the last dose was administered five animals from each group were killed (Group 1) and the following tissues removed for HCB analysis; blood, liver, kidney, fat, heart, lung, spleen, brain, and muscle. Five animals from each group were placed on 25 “/, of their normal food intake for 9 days (Group 3) while the five remaining animals were allowed to feed ad lib. during the same period (Group 2). Urine and feces were collected daily from each animal and on the tenth day after dosing ceased all animals were killed and their tissues removed, weighed, and frozen pending HCB analysis. Tissues were analyzed for HCB content using a glc-EC procedure described earlier (Villeneuve et al., 1974) and results expressed as ppm HCB based on the wet tissue weight. All food consumption, body and tissue weight data were subjected to statistical analysis using Student’s t test. RESULTS None of the animals in the 0, 1.O, or 10 mg/kg HCB groups died as a result of dosing or food restriction. With those animals receiving 100 mg/kg HCB, none died during the 14-day dosing period. However, one animal of this group which was allowed to feed ad lib. subsequent to dosing died 4 days after receiving the last dose of HCB. Of the animals dosed with 100 mg HCBjkg and then placed on 25 % of their normal food intake, four died. Of these, one animal died 3 days after dosing ceased, two rats died 6 days after, and one died 10 days after. In all cases the animals stopped eating 1-2 days prior to the onset of death, and displayed tremors and a reddish nasal discharge. HCB treatment did not affect body weight gain over the 14-day dosing period. The animals which were dosed with HCB at 10 mg/kg and then fed ad lib. (Group 2) gained more weight over the 9-day period than their respective controls. Only animals which received 100 mg/kg showed an increased weight loss when they were placed on 25 % of their normal food intake. Food consumption during the dosing period was not affected by HCB administration. The tissue weights, expressed as percent body weight are shown in Table 1. Food restriction did not affect spleen, brain, kidney, or heart weights but significantly reduced liver weights (p < 0.05) when expressed as a percent of body weight. HCB treatment did not affect brain, kidney, or heart weights at any dose level when expressed as percent body weight. Spleen weight was increased in those animals receiving 100 mg/kg while liver weights were increased at both 10 and 100 mg/kg. In those animals subjected to both HCB and food restriction, increased brain weights (percent body weight) were observed at 10 and 100 mg/kg; increased kidney weights (percent body weight) were observed at 100 mg/kg. HCB administration at both 10 and 100 mg/kg negated the effect of lowered liver/body weights observed during food restriction.
FOOD
RESTRICTION
AND
HCB
1N THE
31.5
RAT
TABLE 1 TISSUE WEIGHTS (%
BODY WEIGHT) OF RATS TREATED HEXACHLOROBENZENE (HCB)
WITH
Heart
Liver
0.32 + 0.01 0.28 k 0.02 0.28 + 0.01
3.59 + 0.08 3.89 + 0.29 2.37 + 0.10
0.31 + 0.01 0.28 + 0.01 0.29 + 0.02
3.48 & 0.06 3.69 + 0.12 2.49 + 0.03
0.75 4 0.02 0.73 * 0.03 0.78 f 0.03
0.32 k 0.01 0.28 k 0.01 0.28 f 0.01
3.97 * o.12c 4.18 t 0.10 4.11 * 0.13’
100 mg/kg HCB 0.44 & 0.02 0.77 2 0.01 0.47 * 0.01 0.80 +_ 0.02 0.67 + 0.03’ 0.89 f 0.02’
0.28 + 0.09 0.28 f 0.02 0.31 + 0.01
5.85 + 0.14’ 6.83 + 0.28’ 6.74 + 0.48’
Groupb
Spleen
Brain
Kidney
1
0.16 + 0.01
2 3
0.15 + 0.01 0.14 + 0.01
0.46 + 0.02 0.45 + 0.03 0.52 + 0.03
1 2 3
0.14 2 0.01 0.14 * 0.01 0.14 * 0.01
0.40 + 0.01 0.55 * 0.02
1 2 3
0.18 f 0.03 0.14 -t 0.02 0.16 + 0.01
0.47 4 0.01 0.46 4 0.03 0.64 + 0.04”
1
0.19 + 0.03’ 0.19 t 0.01’ 0.20 + 0.07’
0 mg/kg HCB 0.72 + 0.02 0.67 f 0.02 0.71 + 0.01
1.Omg/kg HCB 0.50 + 0.01 0.78 + 0.03 0.74 + 0.02 0.73 + 0.02
10 mg/kg HCB
2 3
n Values represent mean t SE of 5 animals. b See text for details of treatment for groups 1, 2, and 3. c Denotes significant difference from 0 group at p i 0.05.
The tissue residue data obtained from the animals dosed 1, 10, and 100 mg/kg is shown in Table 2. In nearly all tissues the concentration of HCB was lower in animals fed ad lib. for 9 days (Group 2) than those killed immediately after dosing (Group 1). Two exceptions to this were fat and muscle tissue. The concentration of HCB was higher in all tissues from animals whose food intake was restricted (Group 3) compared to those fed ad lib. (Group 2). The tissues showing the greatest proportional increase in HCB after food restriction were brain, spleen, liver, and heart followed by fat, plasma, kidney, muscle, and lung. When results were calculated in terms of total micrograms of HCB per tissue, brain again showed the greatest increase foIlowed by liver, spleen, heart, and kidney. All tissues examined accumulated HCB in a dose-dependent manner and levels of residue were always highest in adipose tissue followed by liver, kidney, lung, brain, spleen, heart, muscle, and plasma. The fecal excretion of HCB during the dosing and subsequent food restriction is shown in Fig. 1. The amount of HCB excretion in the first 24 hr for all three doses was approximately 1 “/0 of the amount administered. The excretion profiles were similar for all three levels during the dosing period. Although there was considerable variation in the amount of HCB excreted on a day to day basis, there was no obvious trend toward increased excretion throughout the dosing period. After the last dose of HCB was administered there was a sharp drop in the amount excreted. In the group of animals dosed 1 mg/kg, the animals subjected to food restriction excreted less than the animals fed ad lib. At the 10 and 100 mg/kg levels, the animals subjected to food restriction excreted HCB in amounts similar to those fed ad lib.
20.3 -t 1.4 17.4 * 1.4 191.5 k 25
117+3 83 + 11” 385 + 38
18.9 + 2 13.1 * 0.9 48.2 L- 6
109 + 11 62f6” 309 * 9
2.2 * 0.2 2.8 to.2 9.2 + 1.4
17.9 L- 0.8 12.9 + 1.5’ 43.ge
3.1 F0.4 4.4 +- 0.3 5.5 & 1.4
2.8 + 0.5 1.3 f 0.1 4.1 If: 0.8
0.43 & 0.07 0.31 + 0.01 0.71 to.15
Brain
Spleen
PROFILE
Plasma
RESIDUE
OF
Kidney
177* 11 129 k 11” 467 + 110
175 + 10 90 rt 3d 339 + 26
10mg 34.3 & 2.4 34.5 -t 3.4 46.4 + 5.3 30.2 + 3.5 68.2 _+ 6.3 68.2 _t 6.2 100 mg
-
.-
13.8 f 0.7 20.6 + 6 49.2 + 17 69 &- 6 84 + 18’ 282 f- 170
96 f 2 70 + 3d 297 k 23
2.7 + 0.4 1.2 1.1 2.5 + 0.3 +
Musde
(HCB)
17.2 + 1.4 15.1 * 1.1 66.6 f 12
2.4 k 0.3 1.5 0.1 4.2 f 0.7 +
Heart
HEXACHLOROBENZENE
1.0 mg 4.7 + 0.7 7.7 + 1.0 4.4 -f. 0.3 3.1 + 0.2 1.4 4.8 1.0 6.1 + +
Lung
WITH
HCB (ppm wet weight)
RATS TREATED
2
’ Mean residue concentration &SE of 5 animals except where otherwise indicated. b See text for details of treatment for groups 1, 2, and 3. ’ Mean &SE of 4 animals. d Mean &SE of 3 animals. e Value from I animal only.
Groupb
TISSUE
TABLE
268 t 15 167 + 22” 614 + 70
58.6 & 1.9 35.1 +- 2.0 176 rt 51
5.7 + 0.5 2.9 0.2 7.7 + f 1.4
Liver-
3860 -t 575 3869 + 360’ 10,597’
527 + 32 667 + 61 2204 & 426
87 + 14 59.4 * 4 161 + 6
Fat
is
C
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FIG. 1. Daily fecal excretion mg/animal of HCB during dosing and subsequent period of food restriction. (0-U) 100 mg HCBjkg body wt; (A-A) 10 mg HCB/kg body wt; (x-x) 1 .Omg HCB/kg body wt. The solid line (-) after dosingrepresents animals fed ad lib. The broken line (-- -) represent animals on l/4 normal food intake. Each value during dosing represents the mean daily excretion of 15 animals. Each value after dosing represents the mean daily excretion of 5 animals.
DISCUSSION Hexachlorobenzene has recently gained prominence as an environmental contaminant through its use as a fungicide and an industrial chemical. Like DDT, and other members of the organochlorine group, HCB demonstrates a high persistence,limited biodegradibility, and considerable lipophilicity. Becauseof its structural and chemical similarity to the DDT-type compounds the present study was carried out to determine the extent to which HCB is relocated when fat depots in animals previously exposed to HCB are mobilized. The results in this study confirmed the toxic effects observed in a preliminary study. Toxic signswere observed only in animals dosed 100mg/kg for 14consecutive days. No toxic signswere observed in animals dosed at the other levels except for an increased liver/body weight ratio at 10 mg/kg. Animals which died after receiving 100mg/kg body weight first displayed a lossof appetite, tremors, and a red nasaldischarge.This reddish discharge has been noted in acute DDT poisoning and was attributed to oversecretion
318
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C. VILLENEUVE
of porphyrin by the Harderian glands (Henderson and Woolley, 1970). The combination of HCB administration and food restriction increased spleen weight at 100 mg/kg and increased liver weight at 10 and 100 mg/kg. The latter observation can be related to the ability of HCB to cause liver hypertrophy (Grant et al., 1974). The residue profile indicates that HCB is accumulated in all tissuesexamined in a dose dependent manner. Toxic signsand death were observed in animals whose brain residue levels were in excessof 300 ppm. These results agree with data from the preliminary study where the five animals which died had brain HCB concentrations of approximately 400 ppm while the lone survivor had a brain level of 266 ppm. The one animal which died after being treated with HCB at 100 mg/kg followed by normal food intake had lost 180 g and had a brain concentration of 360 ppm HCB. Fecal analysis indicated that food restriction did not increase the excretion of HCB and thus total body burdens of HCB are not drastically reduced as has been reported for DDT and dieldrin (MacDonald ef al., 1973). Urinary excretion was not monitored in the present study becauseresults from the preliminary experiment indicated that most of the HCB was excreted in the fecesand that the urinary excretion pattern wasessentially the same as for the fecesduring HCB administration and the subsequentperiod of food restriction. The residuedata indicate the concentrations of HCB observed in the brain of dead animals (>300 ppm) are considerably higher than those observed for rats poisoned with DDT [35 ppm (Dale et al., 1962)].
ACKNOWLEDGMENTS The technical assistance of Miss Luz Panopio and Mr. Henry Jamesis gratefully acknowl-
edged. REFERENCES ACKER,
L. AND
SCHULTE,
E. (1970). Appearance of chlorinated biphenylsand hexachloro-
benzene along with insecticides in human milk and fat tissues. Nuturwissenschaften 57, 497. BRADY, M. N. AND SIYALI, D. S. (1972)Hexachlorobenzene in humanbody fat. Med. J. Aust. 1, 158-161. CLAUSEN,
J. AND
KONAT,
G. (1972) Enzymic and behavioral changesinduced in mice fed
polychlorinated biocides followed by starvation. Experientia 28, 902-903. DALE, W. E., GAINES, T. B. and HAYES, W. J. (1963). Storage and excretion of DDT in starved rats. Toxicol. Appl. Pharmacol. 4, 89-106. ECOBICHON, D. J. AND SASCHENBRECKER, P. W. (1969).The redistribution of stored DDT in cockerels under the influence of food deprivation. Toxicol. Appl. Phramacol. 15, 420-432. GRANT, D. L., IVERSON, F., HATINA, G. V. AND VILLENEUVE, D. C. (1974)Effects of hexachlorobenzene on liver porphyrin levels and microsomal enzymes in the rat. Environ. Physiol. Biothem. 4, 159-165. HENDERSON, G. L. AND WOOLLEY, D. E. (1970). Mechanisms of neurotoxic action of 1,1,1trichloro-2.2-bis(p-chlorophenyl) ethane (DDT) in immature and adult rats. J. Pharmacol. Exp. Ther. 175, 60-68. KOEMAN, J. H. TEN NOEUVER DEBRAUUS, AND DE Vos, R. H. (1969). Chlorinated biphenyls in fish mussels and birds from the river Rhine and the Netherlands coastal area. Nature (London) 221, 1126-l 128. MACDONALD, W. E., DEICHMANN, W. B. CUBIT, D. A. AND BEASLEY, A. G. (1973). In Pesticides and the Environment: A Continuing Controversy, pp. 87-101. Intercontinental Book Corporation, New York.
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Report of the Secretary’s Commission on Pesticides and their Relationship to Environmental Health, U.S. Dept. of Health, Education and Welfare, December 1969 (Mrak Report). SIYALI, D. S. (1972). Hexachlorobenzene and other organochlorine pesticides in human blood. Med. J. Artst. 2, 1063-1066. VILLENEUVE, D. C., PHILLIPS, W. E. J., PANOPIO, L. G., MENDOZA, C. E., HATINA, G. V. AND GRANT, D. L. (1974). The effects of phenobarbital and carbon tetrachloride on the rate of decline of body burdens of hexachlorobenzene in the rat. Arch. Environ. Contamitt. Toxicol., 2,243-252. ZABIK, M. E. AND SCHEMMEL, R. (1973). Dieldrin storage of obese, normal and semistarved rats. Arch. Environ. Health 27, 25-30.