6',ancer L,~.tters, 5 (19'~8).91-'~5 © E~h~er/North-Holl[and Scien'~;ificPublishers Ltd.
91
I ] ~ I B I T I O N O F .AZOXYMET.}I[~xNEiNDUC~,D I~T~,ST]NAL CA~[CER B Y DISUII'~B'~AM
NORMA~4 D.,NIGRO i,ndI~,OBERT L. CAMPBELL W ~ y ~ State University Sch~o~'o f Medicine, D,epartment o f Surgery, Detroit. M~chig~n 48261 U.S.A, (Rec.~ived23 Feb~umT 1978) (Revised ,~ersionreceived2~ May 197811 (Accepted 31 May 1978)
S[~M2JZY Sprague--Dawley ra~s, both intact and colostomized .enimals, were ~iven 24 weekly injectious of a~,'oz']methane. Rats '~.:ere f~.d either Rat Pmina Chow or the same diet plus 0.25% d~mlfh'am. I n th~ in~act animals, ~%sulfiram r,.~tuc~ ttunors from an average o f 6 . 3 f~) 0.95. The number of rats developktg ~umo~rs was ~reduced from 100% 1!o 6~% l~ colosb~mized a~,imals, the re:~luc~$)n was from ~ average of 5.0 to 0.13. Marked inhibition o~curmd even k~ the defm~eti[onali[zed colon The results suggest that disuL[~am blocks the metabolism of ~oxymethane ~;omethylazoxymethanol, and ~so that the inhibitor m a y act Systemically.
INTRODUCTION
The ingestionof dJstdfiram has been shown by Wattenbel~g [5] to h~ve a ~h~rong k~hib~toryeffecto,n intestinalcm~inogen~is ~u mice given 1,2 ~lin~ethylhydraz~ae (DMH). At tlm~~,~:~ne the mechanism of ~tion of disulfh'~$a~-as unkno~n. The drug, Wa~enberg poh~tA~ cut, is incomplel~ely absozb,.~,lhu~ its actkm could be ei~herd~rect or systemic. Sub~!.,quently,the metabolism of *iC-lal~eledD M H was studied by ]~al~tet ~d,, [2--4] in rats treated with disulfir~n. Increased amounts of azomethane (AM) and decreased amotmts of ~4CO,~were exh~£,ed by th~se animals. Fmther, decreas,~v/ amounts o f the DMH metabolites azoxymeth~ne (AOMIt and Addresz ,~rrespondence to: Dr. Non, an D. Nigro, Way~.~eSta~e University School of Niedleb~e, Department c~fSurgery, 540 E. Cmffie~i Ave., Detroit, Michigan 4820~l, U.S.A.
Abbrev,ia~ons"AM, azcmethane;AONI, azo~!~me~hane;DMH, 1,2-dlmethylhydr~.,ine; MAM, methyl~,.osym eths~-l.
92
methylazoxymethsnol ( M A M ) were found in the urine. Both of these D M H metabolites are knc~wn intestinalcarcinogens [7]. These observations suggest disul~h'am hlhibitsthe oxidative metabolism of D M H at the A M to:AOM-step, thereby interrupting,,the ~cl,ivationof D M H . W e have previously found a reduced tumor yield in the defunctional.ized portion of the colon in colostomized rats given A O M [1]. This w~)uld sui~ge'.'~t that r~heeffect of t~,~scarcinogen is partly systemic. The objectives of '~he preser.~tstudy with disulfirarnare 2-fold:one, to determine whether itir£hibits tumor formation in rats injected with A O M ; and two, whether ~ts m o d e ol action is local or systemic. MATERIAL A N D METI~IODS Male Sprague--Dawley rats, approx. 8 weeks o f age, were purchased from Spartan Research Anhnals, Inc., Haslett, Michigan. AOM was purchased from Ash Stevens, Inc. Detroit, Michigan and was prepared as an aqueous solution for injection. Disulfiram (i.e.., tetraethylthiuram disulfide) was obtained from AJdrich Chemical Company, Inc. and recrystaUized from ethanol twice to pale yello,: crystals before use. Colostomies were established in t h e midportion o f the large intestinf~ in 50 rats. Twenty-five intact and 25 colostomized animals were fed a normal, Purina rat chow diet. Two s i m i ~ r groups were fed granular Purina rat chow containing 0.25% (w/w) disulfiram Diets were prepared at weekly intervals thtoughoul~ t h e experiment. Each o f these 4 groups received weekly sub,mtaneous injectinn.,~ o f AOM, 8 m g / k g . Two carcinogen-free control g r o u p s o f 20 intact arfimals were e~tablished with i group receiving t h e diet containing disulfiram. All animals were h o r s e d in~.i~idually and allowed their respective diets and water ad libittun. Injections and dietary treatment were continued for the 24-week duration o f the experiment. At t h e end o f the stud~,, t h e suzviving mrs in all groups were sacrificed and necropsies performed. The intestinal tract was opened, and the number, size, and location o f tumors was recozdedo Histological sections were made o f 20 represe~:tat~v~ tumors m a t c h e d as closely as possible as to location and[ size (0.4--1.G ~cm) in the intac ~ animsl! groups. The average n u m b e r o f tumor.~ per animal a~,d t h e standard ezzor (S.E.) were calculated for each group. The data were analyzed by the Student's t-test. ~S~TS
At the end of the study a total of 43 intact arthnalsreceivingA O M remained, 21 fed the normal diet and 22 fed the diet containing disulfiram.I~ the colostomized ar~[malgroups tre~atedwith A O M , only 8 mfimals remained in each " dietary grou~p. T h e 68% lolls in ar~.mals i n these groups was t h e result of complicatio]~ Of the eolostomy and n o t due to t o x i c effects o f treatment with disulfirarn or AGM . . . .
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94 T h e animals accepted the diet conta~.aing d i s u l £ i r ~ , and t h e y cousumed sufficient a m o u n t s so all gained weight to the e n d Of the experiment. However in the disulfiram fed animals, t h e percent.age gain in weight was less as shown in Table 1. The total nttmber o f t u m o r s in the s m ~ t arid large bowel o f each group, as well as the average n u m b e r o f ttmltors per ~at, is shown in Table 1. In t h e intact an~mals~ tbe average total numbe.~ o f t u m o r s was significantly :reduced by disalfiram ( P < 0.0005) from 6.3 -~0.81 to 0L95 ± 0.40. This represents a 6to, 7-~old reduction in t h e n u m b e r o f tumors in both t h e small and large intestine. Flurther, the n u m b e r o f rats developing tumors w~Lsreduced to 60% (13 o u t of 22 rats had tumors} as compared to 100% for t h e animals not on disulfiram. In colostomized rats, df~ulfizam reduced t h e average total n u m b e r o f t u m o r s significantly ( P < 0.005) from 5.0 + 1.3 to 0.13 ± 0.12. In fact, there were no tumors in the small intestine an,~ functional colon in these rats. The n u m b e r o f tumors in t h e defunctionalized colon was reduced by 25-fold as shown in Table 1. There ~vere no intestinal tumors in the 2 animal Stoups n o t receiving AOM. All tumors examined were adenocarcinom;~. The survey showed no difference in the degree of anaplasia or in the penetration o f the intestinal wall between the normal diet and dist~firam fed mrs. DISCUSSION
Our re,mlts clearly indicate that; disulfiram inhibits AOM-induced intestinal t u m o r s i n the rat. Wattenberg [6] recently reported similar zes,lts in the mouse. There was an 87% t u m o r incidence in mice, and a 37% decrease in the m e a n n u m b e r o f tumors. O u r resultS usLug rats a r e more striking, there being a reduction to 60% t u m o r incidence and a n 8 5 % decreasein t h e mean n u m b e r o f tumors. Thus t h e inhibitory effect of ciisulfirmn On A O M carcinogenesis m a y be species dependent. It appears that disulfiram inhibit:; intestina~ caminogenesis with 1,2-DMH or AOM by interrupting the metabolism o f these carcinogens to their active fom~s. Whether this action is direct o r systemic in nature is obviously hnportant. The 25-fold zeductinn in t h e n u m b e r o f t u m o r s present in the defunctionalized colon in the colostomized animals indicates disulfiram has a systemic effect. The effect o f disulfiram o n tumorigenesis m a y also b e due to a depressed nutritional status. However, t h e degree o f inhibition appears far too great to ascribe it to this factor, particularly in view o f t h e inhibitory e f f ~ t s o f disulfiram on the metabolism o f AOM found by Fiala [ 2 ] . Our results combined with those of others suggest disulfiram e~:erts2 blocks in the metabolism 1,2-DMH. The latterblock ( A O M -~ IVIAM) is c o m m o n to the activation of both 1,2,DMH and A O M . Thus a detailed enzym01ogical characterization of this reaction m a Y provide a better understanding of the activation and/or detoxJ.ficationprocesses in relation to :intestinalCm~inogenesis.
9~ B'EFERENCES 1 Campbell, ]~.L., Singh,D.V. and Nigr% N.D. (19~5) ~port~uce of the :~ecalstream o:a the induction of colon tumors by azo~.Tmethane in rat~. Cancer ]~s., :.~6, 1369--1371. 2 Fiata, E.S. (1977) InvesI:~'ations ~nto the metabolism and mode of a~ti~n of the co]Ion earcinogem~ 1,2-dimethylhydLrazi~teand azoxymethane. Cancer,.40, 24~6--24~5. 3 Fiala, E S Bobota~ G , Ku[vki~ C , Wattenberg. L W and Weh'ber~er J H 1977t Effects of c lsulfiram and lmlated compounds on the metabolism i~1vlvo c.f the colon carcinogens, 1 ;2.dimethylhydrazin,~,. Bi~che~. PharmacoL, 26,1763--1768. 4 Fiala, E.S., Bobota~, G. Kt~lakis, C. znd Weisburger, J.H. (19771b Inhibition of 1,2d~methylhydrazine met~ol~a~ by dlst~lfiram. Xenobiotica, 7, 5--9. 5 Wattenbvzg, L.W.(1975)li~hibitionofdimethylhydr~sine.~nduced neophmiao£thelm.~e int~tine b~f distflfiram. J. N~ttl. Ca~RcerInst., 54, 1005--1006. 6 Wattenberg, L.W., Lain, L•K.T., Fl~dmore, A.V. and Borc~ert, P. (1977) ~nhibitor~ o~ colon carcir~ogenesls. Czncer, 40, 2d~32-2435. 7 Weisburger, J.H, Reddy, B.S. and Wynder, E.L. (1977) Colon cancer: its epidemiolog~ and experi~nental production. Canc~r, ~i0, 2414--2420.
91
I ] ~ I B I T I O N O F .AZOXYMET.}I[~xNEiNDUC~,D I~T~,ST]NAL CA~[CER B Y DISUII'~B'~AM
NORMA~4 D.,NIGRO i,ndI~,OBERT L. CAMPBELL W ~ y ~ State University Sch~o~'o f Medicine, D,epartment o f Surgery, Detroit. M~chig~n 48261 U.S.A, (Rec.~ived23 Feb~umT 1978) (Revised ,~ersionreceived2~ May 197811 (Accepted 31 May 1978)
S[~M2JZY Sprague--Dawley ra~s, both intact and colostomized .enimals, were ~iven 24 weekly injectious of a~,'oz']methane. Rats '~.:ere f~.d either Rat Pmina Chow or the same diet plus 0.25% d~mlfh'am. I n th~ in~act animals, ~%sulfiram r,.~tuc~ ttunors from an average o f 6 . 3 f~) 0.95. The number of rats developktg ~umo~rs was ~reduced from 100% 1!o 6~% l~ colosb~mized a~,imals, the re:~luc~$)n was from ~ average of 5.0 to 0.13. Marked inhibition o~curmd even k~ the defm~eti[onali[zed colon The results suggest that disuL[~am blocks the metabolism of ~oxymethane ~;omethylazoxymethanol, and ~so that the inhibitor m a y act Systemically.
INTRODUCTION
The ingestionof dJstdfiram has been shown by Wattenbel~g [5] to h~ve a ~h~rong k~hib~toryeffecto,n intestinalcm~inogen~is ~u mice given 1,2 ~lin~ethylhydraz~ae (DMH). At tlm~~,~:~ne the mechanism of ~tion of disulfh'~$a~-as unkno~n. The drug, Wa~enberg poh~tA~ cut, is incomplel~ely absozb,.~,lhu~ its actkm could be ei~herd~rect or systemic. Sub~!.,quently,the metabolism of *iC-lal~eledD M H was studied by ]~al~tet ~d,, [2--4] in rats treated with disulfir~n. Increased amounts of azomethane (AM) and decreased amotmts of ~4CO,~were exh~£,ed by th~se animals. Fmther, decreas,~v/ amounts o f the DMH metabolites azoxymeth~ne (AOMIt and Addresz ,~rrespondence to: Dr. Non, an D. Nigro, Way~.~eSta~e University School of Niedleb~e, Department c~fSurgery, 540 E. Cmffie~i Ave., Detroit, Michigan 4820~l, U.S.A.
Abbrev,ia~ons"AM, azcmethane;AONI, azo~!~me~hane;DMH, 1,2-dlmethylhydr~.,ine; MAM, methyl~,.osym eths~-l.
92
methylazoxymethsnol ( M A M ) were found in the urine. Both of these D M H metabolites are knc~wn intestinalcarcinogens [7]. These observations suggest disul~h'am hlhibitsthe oxidative metabolism of D M H at the A M to:AOM-step, thereby interrupting,,the ~cl,ivationof D M H . W e have previously found a reduced tumor yield in the defunctional.ized portion of the colon in colostomized rats given A O M [1]. This w~)uld sui~ge'.'~t that r~heeffect of t~,~scarcinogen is partly systemic. The objectives of '~he preser.~tstudy with disulfirarnare 2-fold:one, to determine whether itir£hibits tumor formation in rats injected with A O M ; and two, whether ~ts m o d e ol action is local or systemic. MATERIAL A N D METI~IODS Male Sprague--Dawley rats, approx. 8 weeks o f age, were purchased from Spartan Research Anhnals, Inc., Haslett, Michigan. AOM was purchased from Ash Stevens, Inc. Detroit, Michigan and was prepared as an aqueous solution for injection. Disulfiram (i.e.., tetraethylthiuram disulfide) was obtained from AJdrich Chemical Company, Inc. and recrystaUized from ethanol twice to pale yello,: crystals before use. Colostomies were established in t h e midportion o f the large intestinf~ in 50 rats. Twenty-five intact and 25 colostomized animals were fed a normal, Purina rat chow diet. Two s i m i ~ r groups were fed granular Purina rat chow containing 0.25% (w/w) disulfiram Diets were prepared at weekly intervals thtoughoul~ t h e experiment. Each o f these 4 groups received weekly sub,mtaneous injectinn.,~ o f AOM, 8 m g / k g . Two carcinogen-free control g r o u p s o f 20 intact arfimals were e~tablished with i group receiving t h e diet containing disulfiram. All animals were h o r s e d in~.i~idually and allowed their respective diets and water ad libittun. Injections and dietary treatment were continued for the 24-week duration o f the experiment. At t h e end o f the stud~,, t h e suzviving mrs in all groups were sacrificed and necropsies performed. The intestinal tract was opened, and the number, size, and location o f tumors was recozdedo Histological sections were made o f 20 represe~:tat~v~ tumors m a t c h e d as closely as possible as to location and[ size (0.4--1.G ~cm) in the intac ~ animsl! groups. The average n u m b e r o f tumor.~ per animal a~,d t h e standard ezzor (S.E.) were calculated for each group. The data were analyzed by the Student's t-test. ~S~TS
At the end of the study a total of 43 intact arthnalsreceivingA O M remained, 21 fed the normal diet and 22 fed the diet containing disulfiram.I~ the colostomized ar~[malgroups tre~atedwith A O M , only 8 mfimals remained in each " dietary grou~p. T h e 68% lolls in ar~.mals i n these groups was t h e result of complicatio]~ Of the eolostomy and n o t due to t o x i c effects o f treatment with disulfirarn or AGM . . . .
i , ! ii i !
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~
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~ l~ 0
94 T h e animals accepted the diet conta~.aing d i s u l £ i r ~ , and t h e y cousumed sufficient a m o u n t s so all gained weight to the e n d Of the experiment. However in the disulfiram fed animals, t h e percent.age gain in weight was less as shown in Table 1. The total nttmber o f t u m o r s in the s m ~ t arid large bowel o f each group, as well as the average n u m b e r o f ttmltors per ~at, is shown in Table 1. In t h e intact an~mals~ tbe average total numbe.~ o f t u m o r s was significantly :reduced by disalfiram ( P < 0.0005) from 6.3 -~0.81 to 0L95 ± 0.40. This represents a 6to, 7-~old reduction in t h e n u m b e r o f tumors in both t h e small and large intestine. Flurther, the n u m b e r o f rats developing tumors w~Lsreduced to 60% (13 o u t of 22 rats had tumors} as compared to 100% for t h e animals not on disulfiram. In colostomized rats, df~ulfizam reduced t h e average total n u m b e r o f t u m o r s significantly ( P < 0.005) from 5.0 + 1.3 to 0.13 ± 0.12. In fact, there were no tumors in the small intestine an,~ functional colon in these rats. The n u m b e r o f tumors in t h e defunctionalized colon was reduced by 25-fold as shown in Table 1. There ~vere no intestinal tumors in the 2 animal Stoups n o t receiving AOM. All tumors examined were adenocarcinom;~. The survey showed no difference in the degree of anaplasia or in the penetration o f the intestinal wall between the normal diet and dist~firam fed mrs. DISCUSSION
Our re,mlts clearly indicate that; disulfiram inhibits AOM-induced intestinal t u m o r s i n the rat. Wattenberg [6] recently reported similar zes,lts in the mouse. There was an 87% t u m o r incidence in mice, and a 37% decrease in the m e a n n u m b e r o f tumors. O u r resultS usLug rats a r e more striking, there being a reduction to 60% t u m o r incidence and a n 8 5 % decreasein t h e mean n u m b e r o f tumors. Thus t h e inhibitory effect of ciisulfirmn On A O M carcinogenesis m a y be species dependent. It appears that disulfiram inhibit:; intestina~ caminogenesis with 1,2-DMH or AOM by interrupting the metabolism o f these carcinogens to their active fom~s. Whether this action is direct o r systemic in nature is obviously hnportant. The 25-fold zeductinn in t h e n u m b e r o f t u m o r s present in the defunctionalized colon in the colostomized animals indicates disulfiram has a systemic effect. The effect o f disulfiram o n tumorigenesis m a y also b e due to a depressed nutritional status. However, t h e degree o f inhibition appears far too great to ascribe it to this factor, particularly in view o f t h e inhibitory e f f ~ t s o f disulfiram on the metabolism o f AOM found by Fiala [ 2 ] . Our results combined with those of others suggest disulfiram e~:erts2 blocks in the metabolism 1,2-DMH. The latterblock ( A O M -~ IVIAM) is c o m m o n to the activation of both 1,2,DMH and A O M . Thus a detailed enzym01ogical characterization of this reaction m a Y provide a better understanding of the activation and/or detoxJ.ficationprocesses in relation to :intestinalCm~inogenesis.
9~ B'EFERENCES 1 Campbell, ]~.L., Singh,D.V. and Nigr% N.D. (19~5) ~port~uce of the :~ecalstream o:a the induction of colon tumors by azo~.Tmethane in rat~. Cancer ]~s., :.~6, 1369--1371. 2 Fiata, E.S. (1977) InvesI:~'ations ~nto the metabolism and mode of a~ti~n of the co]Ion earcinogem~ 1,2-dimethylhydLrazi~teand azoxymethane. Cancer,.40, 24~6--24~5. 3 Fiala, E S Bobota~ G , Ku[vki~ C , Wattenberg. L W and Weh'ber~er J H 1977t Effects of c lsulfiram and lmlated compounds on the metabolism i~1vlvo c.f the colon carcinogens, 1 ;2.dimethylhydrazin,~,. Bi~che~. PharmacoL, 26,1763--1768. 4 Fiala, E.S., Bobota~, G. Kt~lakis, C. znd Weisburger, J.H. (19771b Inhibition of 1,2d~methylhydrazine met~ol~a~ by dlst~lfiram. Xenobiotica, 7, 5--9. 5 Wattenbvzg, L.W.(1975)li~hibitionofdimethylhydr~sine.~nduced neophmiao£thelm.~e int~tine b~f distflfiram. J. N~ttl. Ca~RcerInst., 54, 1005--1006. 6 Wattenberg, L.W., Lain, L•K.T., Fl~dmore, A.V. and Borc~ert, P. (1977) ~nhibitor~ o~ colon carcir~ogenesls. Czncer, 40, 2d~32-2435. 7 Weisburger, J.H, Reddy, B.S. and Wynder, E.L. (1977) Colon cancer: its epidemiolog~ and experi~nental production. Canc~r, ~i0, 2414--2420.
