Modification of hexachlorophene toxicity by dieldrin and Aroclor 1254

Toxicology, 2 (1974) 309--318 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands

MODIFICATION OF HEXACHLOROPHENE AND AROCLOR 1254

T O X I C I T Y BY D I E L D R I N

DAVID C. L. JONES, WILLIAM E. DAVIS JR., GORDON W. NEWELL, DANIEL P. SASMORE and VICTOR J. ROSEN* Life Sciences Division, Stanford Research Institute, Menlo Park, Calif. 94025, and *Department of Pathology, Memorial Hospital of Southern California, Culver City, Calif. 90230 (U.S.A.)

(Received March 4th, 1974) (Accepted March 5th, 1974)

SUMMARY H e x a c h l o r o p h e n e (HCP) was fed singly or paired w i t h either a f l a t o x i n B 1 ( A F ) , A r o c l o r 1 2 5 4 ( A R ) , or dieldrin (D) t o F i s c h e r - 3 4 4 rats f o r 8 w e e k s b e g i n n i n g at 5 w e e k s o f age. F o r H C P fed singly, t h e r e was n o m o r t a l i t y at 4 0 0 p p m or less, 80% m o r t a l i t y in m a l e s a n d 73% in f e m a l e s at 600 p p m , a n d 1 0 0 % m o r t a l i t y in b o t h sexes at 8 2 0 p p m . Paralysis was a p p a r e n t in o n e f e m a l e at 4 0 0 p p m a n d in all animals at 6 0 0 p p m . Minimal n e u r o n a l d e g e n e r a t i o n was e v i d e n t in t h e brain at 50 p p m , focal necrosis and m y e l i n v a c u o l i z a t i o n at 4 0 0 p p m or higher, a n d f o c a l h e m o r r h a g e at 6 0 0 p p m . While a d d i t i o n o f A F at 1 p p m did n o t a f f e c t t h e t o x i c i t y of 6 0 0 p p m o f HCP, a d d i t i o n o f A R at 160 p p m r e d u c e d m o r t a l i t y t o less t h a n 7%, a n d a d d i t i o n o f D at 100 p p m to a d i e t c o n t a i n i n g 9 0 0 p p m o f H C P r e d u c e d m o r t a l i t y f r o m 1 0 0 % to less t h a n 4%. F o r b o t h t h e H C P / A R a n d t h e H C P / D c o m b i n a tions, n o paralysis was o b s e r v e d , a l t h o u g h t h e histologic changes in t h e brain w e r e still a p p a r e n t .

INTRODUCTION A l t h o u g h t h e t o x i c i t y o f H C P has b e e n r e p o r t e d f o r a v a r i e t y o f species, i n c l u d i n g m a n [ 1 ] , o n l y a f e w studies h a v e i n v e s t i g a t e d its t o x i c i t y w h e n t h e Abbreviations: AF, aflatoxin B1 (metabolite of AspergiUus flavus Link ex Fries; purity > 98%); AR, Aroclor 1254 (mixture of polychlorinated biphenyls containing 54% chlorine; Monsanto Chem. Co., St. Louis, Mo.); D, dieldrin (1,2,3,4,10,10-hexachloro-6,7-epoxy-1, 4,4a,5,6,7,8,8a, octahydro-l,4,5,8-dimethanonaphthalene; Shell Chem. Co., Modesto, Calif.; purity > 99%); HCP, hexachlorophene (2,2'-methylene-bis (3,4,6-trichlorophenol); Givaudan Corp., Clifton, N.J.;purity > 99%.

309

compound was fed to laboratory animals over an extended period. Kimbrough and Gaines [2] reported paralysis, increased brain weight and histologic changes in brain tissue of Sherman rats fed a diet containing 500 ppm of HCP for 10--14 weeks, but observed no mortality within 8 weeks. Nakaue e t al. [3] fed HCP at levels ranging from 12.5 to 400 ppm to Wistar rats for 16 weeks, and found complete mortality within 10 days at 400 ppm; there was transitory paralysis and growth depression at 200 ppm, and histologic changes in brain tissue at 100 or 200 ppm but no histologic changes in the brain at 50 ppm or less. They concluded that a minimum " n o effect" level of HCP was between 3.7 and 7.7 mg/kg/day. Recently, Towfighi e t al. [4] reported similar pathologic changes, both in the central nervous system and in peripheral nerve, in Osborne-Mendel rats fed either 300 or 1000 ppm of HCP for up to 8 weeks, paralysis at both dietary levels and some mortality at the higher one. As part of an extensive study of tumorigenesis in rats fed various compounds and pairs of compounds, HCP was fed singly and paired with AF, or AR. After 8 weeks, surviving animals were sacrificed and brain tissues evaluated histologically. Using criteria of 8-week mortality, body weight and neurologic evidence, a marked reduction in the toxicity of HCP was observed when it was paired with either D or AR, but n o t when it was paired with AF. MATERIALS AND METHODS Male and female Fischer-344 rats were received from the supplier (Simonsen Laboratories, Gilroy, Calif.) at 4 weeks of age. They were housed (5/cage) in conventional plastic cages with filter tops, and supplied low-fat Purina Lab Chow (meal) supplemented with refined corn oil (Staley Mfg. Co., Orange, Calif.) and water ad l i b i t u m for 1 week. At 5 weeks of age, cages of rats were distributed among the various control and experimental groups, and feeding of test diets commenced. Test chemicals were analyzed for purity and for stability in diet prior to use. The chemicals were either dispersed in the corn oil as a vehicle prior to incorporation in the diet or, in some cases, mixed in the diet prior to adding the oil. Total fat content of the mixed diet was 6.5%. Animals and food containers were weighed weekly and consumption of chemicals was estimated from container--weight differences and diet concentration for each cage. Spillage from the wire-screened containers was negligible, and the estimates are considered reliable when mortality was not appreciable. During .weeks where significant mortality occurred, reliability of food consumption and chemical dose estimates was less because of the decreasing food consumption of moribund animals. Cages were checked daily for mortality. At the end of 8 weeks surviving animals were chloroformed and tissues taken for histologic evaluation using formalin fixative and conventional staining procedures.

310

RESULTS The data for animals fed HCP singly at levels of 200 to 820 ppm are summarized in Table I. There was no mortality at 400 ppm or less, but 80% o f the males and 73% of the females succumbed within 5 weeks at 600 ppm , with most of the deaths occurring in the first 3 weeks. At 820 ppm all animals died within 2 weeks. B ody weight was essentially unaffect ed by the 2 0 0 p p m diet, but there was a consistent weight d e c r e m e n t compared to controls at levels of 400 ppm or higher. Terminal weight decrements appeared to be a f unc t i on of diet level. Paralysis was n o t observed at 200 ppm, b u t was apparent in one female at 400 ppm and in all animals at 600 ppm beginning in the second week. No paralysis was observed at 820 ppm, presumably because of the early mortality. Histologically, minimal neuronal degeneration in the brain was f o u n d at 5 0 , 1 0 0 and 200 ppm, focal necrosis and myelin vacuolization in all animals at 400 ppm, and additional focal hemorrhage at 600 ppm. No histologic evaluation was made at 820 ppm. The results for animals fed AF, AR or D are shown in Table II. These data are for groups fed single c o m p o u n d s at or near the levels used in the combination studies to be presented. No mortality was observed for any of the groups shown in Table II. In ot her experiments, we have f o u n d no 8-week mo r tality f or AF fed at 4.5 ppm, 27% mortality in males and 7% in females fed AR at 400 ppm, and 20% mortality in males and 13% in females fed D at 150 ppm. Thus, the levels of the 3 c o m p o u n d s used in the present study are clearly in the sublethal or low-lethal ranges when fed singly. From Table II it appears th at AF at 1 ppm , and AR at 200 ppm elicit b o d y weight decrements relative to controls, but t hat D at 100 ppm does not. No paralysis was observed for any of these c o m p o u n d s fed singly. Focal neuronal necrosis was observed in almost all animals fed D at 100 ppm, but no effect of the other 2 c o m p o u n d s u p o n brain histology was apparent. Data for the diets combining HCP with one of the ot her c o m p o u n d s are summarized in Table III. By comparison with the data in Table I, it appears that the addition of AF at I ppm to a diet containing 600 ppm of HCP results in at least as great an 8-week m ort al i t y as with the latter c o m p o u n d fed singly at the same concentration. It is even possible that there is some additivity of these 2 c o m p o u n d s , but the present dietary levels are n o t suitable for evaluating this possibility. Paralysis and brain pathology were similar to those of the animals fed 600 ppm of HCP alone. The addition of 160 ppm of AR to a diet containing 600 ppm of HCP reduced 8-week m or t a l i t y f r om over 70% to less than 7%. Body weights were still less than in controls, b u t weight gains during the last 4 weeks were greater than with the HCP alone. The reduced t o x i c i t y apparently was n o t mediated through reduced c o n s u m p t i o n of the ttCP, as shown by the f o o d c o n s u m p t i o n and c o m p u t e d dose data. The decrease in m or t al i t y when 100 p p m of D was added to a diet containing 900 p p m of HCP (Table III) is even m ore dramatic, since t hat level of HCP is well in excess of the 820 ppm that, fed singly, p r o d u c e d 100%

311

~0

bO 0

0 0

0 0

0 0 ~"

t~

t"'Y

O O

N

O [,-2

2:

Z ;>

N

O Z <

~0

> tn

C~

15 15 15 15 15 15 15 15 15

0 1 2 3 4 5 6 7 8

1

AF

81 92 101 118 140 159 171 179 187

84 102 126 146 170 195 207 219 237 57 87 93 98 105 119 109 111

83 104 115 118 116 130 130 122

Food (g/rat/wk)

0.10 0.13 0.12 0.11 0.10 0.10 0.09 0.09

Chem. dose (mg/kg/day)

15 15 15 15 15 15 15 15 15

60 60 60 60 60 60 60 60 60

Number o f rats

Consumption

Number o f rats

Body weight (g)

Female

Male

60 60 60 60 60 60 60 60 60

Week

0 1 2 3 4 5 6 7 8

Diet level (ppm)

Ctrl

Chem.

73 84 95 106 113 122 128 133 138

73 85 99 110 119 133 141 146 156

Body weight (g)

55 75 75 79 80 82 81 79

67 83 84 83 77 85 85 82

Food (g/rat/wk)

Consumption

0.10 0.12 0.11 0.10 0.10 0.09 0.09 0.08

Chem. dose (mg/kg/day)

M O R T A L I T Y , BODY WEIGHT, F O O D C O N S U M P T I O N AND DOSE IN F I S C H E R - 3 4 4 R A T S F E D AF, A R OR D F O R 8 W E E K S

T A B L E II

0

314

O 0

0 O r~

ba

15 14 5 0

0 1 2 3 4

600 1

HCP + A F

77 67 73

86 106 130 155 171 189 199 217 229 46 47

84 101 107 117 123 117 126 128

Food (g/rat/wk)

54 58

HCP

0.09 0.10

Other

Dose (mg/kg/day)

15 14 5 4 0

6O 60 60 60 60 60 60 60 60

Number o f rats

Consumption

Number o f rats

Body weight (g)

Female

Male

60 60 60 60 60 60 60 60 60

Week

0 1 2 3 4 5 6 7 8

Diet level (ppm)

Control

Chem.

65 63 74 85

75 93 106 117 129 135 141 150 153

Body weight (g)

43 46 54

73 82 77 88 87 84 85 86

Food (g/rat/wk)

57 60 59

HCP

0.10 0.10 0.10

Other

Dose (mg/kg/day)

Consumption

MORTALITY, BODY WEIGHT, FOOD CONSUMPTION AND DOSE IN FISCHER-344 RATS FED HCP IN COMBINATION WITH EITHER AF, AR OR D FOR 8 WEEKS

TABLE III

~0

o~

Diet level (ppm)

600 160

900 100

Chem.

HCP + AR

HCP + D

T A B L E III ( c o n t i n u e d )

0 1 2 3 4 5 6 7 8

0 1 2 3 4 5 6 7 8

Week

30 30 29 29 29 29 29 29 29

30 30 30 29 29 29 28 28 28 93 89 102 113 125 143 158 164 177

79 84 92 104 116 130 147 155 165 59 79 89 97 103 111 114 118

61 75 81 92 101 106 98 107

Food (g/rat/wk)

83 107 106 105 99 95 91 89

64 74 71 72 71 66 55 57

HCP

9 12 12 12 11 11 10 10

17 20 19 19 19 18 15 15

Other

Dose (mg/kg/day)

30 30 30 30 30 30 30 30 30

30 30 30 30 30 30 30 30 30

Number o f rats

Consumption

Number of rats

Body weight (g)

Female

Male

80 75 83 88 94 102 110 114 121

67 69 76 80 85 91 98 102 104

Body weight (g)

50 65 68 73 74 77 79 79

51 61 64 68 68 68 58 65

Food (g/rat/wk)

83 106 103 103 97 94 91 87

64 72 70 71 66 62 50 54

HCP

9 12 11 11 11 10 10 10

17 19 19 19 18 16 13 14

Other

Dose (mg/kg/day)

Consumption

m o r t a l i t y w i t h i n 2 w e e k s ( T a b l e I). A g a i n , t h e e f f e c t was n o t m e d i a t e d through reduced consumption of the chemical. For both the HCP/D and the H C P / A R c o m b i n a t i o n s , t h e r e was n o p a r a l y s i s o b s e r v e d , b u t t h e r e was n e u r o n a l d e g e n e r a t i o n a n d m y e l i n v a c u o l i z a t i o n in all a n i m a l s . DISCUSSION F o r ease o f c o m p a r i s o n , p r e v i o u s l y r e p o r t e d HCP t o x i c i t y d a t a h a v e b e e n s u m m a r i z e d in T a b l e IV w i t h t h o s e o f t h e p r e s e n t s t u d y , using c r i t e r i a o f 8 - w e e k m o r t a l i t y , p a r a l y s i s a n d h i s t o l o g i c c h a n g e s in t h e n e r v o u s s y s t e m . F r o m T a b l e IV it is a p p a r e n t t h a t t h e j u v e n i l e r a t s o f N a k a u e e t al. [3] w e r e t h e m o s t s e n s i t i v e in t e r m s o f m o r t a l i t y a n d p a r a l y s i s . T h e j u v e n i l e r a t s o f t h e p r e s e n t s t u d y w e r e t h e n e x t m o s t s e n s i t i v e in t e r m s of m o r t a l i t y b u t were relatively resistant from the s t a n d p o i n t of paralysis. The adult rats of T o w f i g h i e t al. [4] w e r e h i g h l y r e s i s t a n t in t e r m s o f m o r t a l i t y a n d r e l a t i v e l y r e s i s t a n t on t h e basis o f p a r a l y s i s . T h e a d u l t r a t s o f K i m b r o u g h a n d G a i n e s [2] w e r e also r e s i s t a n t in t e r m s o f m o r t a l i t y , b u t n o m o r e s p e c i f i c i n t e r p r e t a t i o n o f t h e i r f i n d i n g s is p o s s i b l e in t h e p r e s e n t c o n t e x t b e c a u s e t h e y r e p o r t e d r e s u l t s f o r o n l y o n e d i e t level. F o r t h e t w o s t u d i e s i n v o l v i n g m o r e t h a n t w o d i e t a r y levels o f HCP, his. t o l o g i c c h a n g e s in t h e n e r v o u s s y s t e m w e r e a p p a r e n t at HCP d i e t a r y levels well b e l o w t h o s e e l i c i t i n g p a r a l y s i s a n d in t h e 2 - d o s e s t u d y m o s t o f t h e

TABLE IV EXTENT OF MORTALITY, CLINICAL SYMPTOMS (PARALYSIS) AND HISTOPATHOLOGIC CHANGES (NERVOUS SYSTEM) IN RATS RECEIVING HCP IN THE DIET FOR 8 WEEKS Nakaue et

Strain Sex Age (week 0) Number of rats/cage Total rats/group Diet levels (ppm) Mortality (%-- ppm) Paralysis (%-- ppm) Histopathology

al. [3]

Present study

Wistar M and F 4 weeks 1 24 12.5 -- 400 0 -- 200 100 - - 4 0 0 0 -- 100 21 b -- 200 100 - - 4 0 0 0 -- 50

Fischer-344 Sherman M and F F 5 weeks Adult 5 1 20 or 30 22 50 -- 820 500 0 -- 400 0 -- 500 90--600 0 - - 200 > 8 0 - - 500 3 -- 400 100--600 1 0 0 - - 50 100--500

100

--

100

Kimbrough and Gaines [2]

Towfighi et al. [4]

Osborne-Mendel M and F Adult (200 g) Not specified 20 300, 1000 0 -- 300 "few" - - 1 0 0 0 "few" -- 300 100 a -- 1000 "most"-100

a

--

300 1000

a Estimated from text statement. b Transient paralysis.

317

a n i m a l s at t h e l o w e r level s h o w e d histologic changes while f e w o f t h e m e x h i b i t e d paralysis. Thus, while d i f f e r e n c e s in e x p e r i m e n t a l p r o c e d u r e m a y also have b e e n a f a c t o r , t h e d a t a s u m m a r i z e d in T a b l e IV i n d i c a t e t h a t t h e r e are wide ranges in sensitivity to H C P a m o n g strains and ages o f rats, p a r t i c u l a r l y in t e r m s o f m o r t a l i t y , b u t t h a t , regardless o f age or strain, the histologic changes in the n e r v o u s s y s t e m are evident at l o w e r d i e t a r y levels o f H C P t h a n are p a r a l y t i c s y m p t o m s , and t h a t the latter a p p e a r at l o w e r d i e t a r y levels t h a n t h o s e producing mortality. T o o u r k n o w l e d g e , a m e l i o r a t i o n o f the t o x i c e f f e c t s o f HCP b y simultan e o u s a d m i n i s t r a t i o n o f o t h e r c h e m i c a l s has n o t b e e n p r e v i o u s l y r e p o r t e d . H o w e v e r , since b o t h D and A R have b e e n s h o w n to be c a p a b l e o f m o d i f y i n g the toxic and/or pharmaceutical effects of other chlorinated hydrocarbons [ 5 - - 7 ] , a p p a r e n t l y t h r o u g h the m e c h a n i s m o f e n z y m e i n d u c t i o n [ 8 ] , it is r e a s o n a b l e to a s s u m e t h a t a similar m e c h a n i s m underlies t h e i r e f f e c t o n H C P toxicity. S o m e c a u t i o n m u s t be used in i n t e r p r e t i n g the p r e s e n t results. T h e p r e s e n t evidence p e r t a i n s o n l y to 8 - w e e k m o r t a l i t y . While t h e o b s e r v e d b o d y weight and f o o d c o n s u m p t i o n p a t t e r n s o f rats fed HCP in c o m b i n a t i o n w i t h D or A R do n o t i m p l y i m m i n e n t d e a t h a f t e r 8 weeks, t h e histologic evidence indicates t h a t i n t e r a c t i o n was n o t s u f f i c i e n t l y s t r o n g to p r e v e n t p a t h o l o g i c changes in the brain, a n d n o c o n c l u s i o n s relative t o t h e u l t i m a t e m o r t a l i t y o f these animals are w a r r a n t e d . ACKNOWLEDGEMENTS T h e research u p o n w h i c h this p u b l i c a t i o n is b a s e d was p e r f o r m e d p u r s u a n t to C o n t r a c t No. N I H - 7 1 - 2 1 1 6 w i t h the N a t i o n a l I n s t i t u t e o f Health, U. S. D e p a r t m e n t o f H e a l t h , E d u c a t i o n a n d Welfare a n d a c c o r d i n g t o the principles e n u n c i a t e d in t h e " G u i d e f o r L a b o r a t o r y A n i m a l Facilities and C a r e " p r e p a r e d b y t h e N a t i o n a l A c a d e m y o f Sciences - - N a t i o n a l R e s e a r c h Council. T h e a u t h o r s wish to a c k n o w l e d g e the t e c h n i c a l assistance o f M. J. Hylk e m a , D.E. Pratt, C.J. R u s h b r o o k and t h e i r assistants. REFERENCES 1 2 3 4 5 6

R. D. Kimbrough, Arch. Environ. Health, 23 (1971) 119. R. D. Kimbrough and T. B. Gaines, Arch. Environ. Health, 23 (1971) 114. H. S. Nakaue, F. N. Dost and D. R. Buhler, Toxicol. Appl. Pharmacol., 24 (1971) 239. J. Towfighi, N. K. Gonatas and L. McCree, Lab. Invest., 29 (1973) 428. A. tI. Conney, Pharmacol. Rev., 19 (1967) 317. D. R. Bickers, L. C. Harber, A. Kappas and A. P. Alvares, Res. Commun. Chem. Pathol. Pharmacol., 3 (1972) 505. 7 H. Popper, P. Czygan, H. Greim, F. Schaffner and A. J. Garro, Proc. Soc. Exptl. Biol. Med., 142 (1973) 727. 8 R. R. Brown, J. A. Miller and E. C. Miller, J. Biol. Chem., 209 (1954) 211.

318