Cancer Letters, 5 (1978) 173--178 c.~, E,.Izewer/North-t'IollandScientific Fubhshezs ]Ltd.
173
2.ACETYLAM~b~OFLUOEE} ~E-~NDUC~D DNSC~',DULED ])I~A SYNTHE,%~S L'~.~ P A T O C Y T ~ , S I S O L A T E D FI~OM 3 . M F . T H Y L C H O L A b r r H ~ E N E TREA'J?ED I~ATS ]).A.CASCIANO and J.A.F A R R Department o f Health, ]3'd~eat~on c,n d Welfore, ]~bod and Drug Adm~ni~tra~ion, *Nati~)md Center for Toxicolo~cal R~seareh, Jefferson, Arhan~as 72079 (U.S.A.)
J.W. OLDHAM[a~d M.D. CAVE The Unfwersity of Arhanaas for Medical ~,c.~ences, Graduate Program for il~terdisciplinary 'Fox~eology, D~,partmen¢ o f Anatomy 456'1 W, Marh~am, Little Roch, Arhcnsas 7220~ IU, S.A )
(Received 11 hd[ay1973) (AeeepCed 2 June 1978)
The dose-dependent induction of unsehedu]ed DNA synthesis (lJI~S) by 2.acety']~ninofluorene (AAF) and N-OH-2-ace~y}amino:{]uerene (N.GI'I AAF) ~,~primary rat hepatocytes iso,Latedfrom un~'eated and 3-methyJcholantlu'e.lle (3-MC) treated rats was invesl,lgated. 3-MC treatment w~as not necessary for ~he dose-dependent induction of U D S fmduced by N - O H A A F , sug~e:~tingthe presence of enzyme levels adequate for its estenfication to ~m active form in il;olated primary hepatocy~s. Although all doses of AAF increased the level of [SH][thymid£ae incc,rporationabove tha~ of ~he control, a dose-dependenl~ response could not be demonstrated, suggesting inadequate constitutivelevels of N-hydroxyla[,~ng enzymes. Treatment of rats with 3-MC 24 h prier to hepatocyte isolation:~sulted ia a dose-dependent induction of U D S b'.¢AAF. ..MC treatment increased the amount of U D S per cell,as wel~ as the percentage ef cellsinduced to repairtheir D N A .
IN T RO]'},UCTION
The use of the primary ra~ hepatoq~,te cuRure in col~junetmn wi~';hthe Address correspondence to D.~. Casclano, ~n.D , DlL~lmonof Mutagenesls Research, I-IF'~120, Natlona! Center for Toxicological Research, Jefferson, Arkansas 7207~.), qJ.SA Ab~.rev~itionJ~. AAF, 2-ace~ylmI~~ofluorene;DMSO, dhrne~yl su~foxltde;HBSSo Bank's
ba]ta.acedsaltsolution;~-It~C,.bmethylcholanthrene,N-OH AFF, N-OH-2-aeetylaminoflaol,'ene;UDS, um~,cheduledD N A synthesls;UV, ultraviolet
174 induction of UD,S as a predictive system for potential carcinogenic activity o f a ehemk:al compound was first suggested by Williams [10,11]. The hl,epatocyte not~ only serves as the target for DNA dmnage and its subsequent repair by a given chemical, but also contains constitutive levels of enzymes necessm'y for activa~,ion o f p~ecareinogens to ~heir reactive form. The system is therefo~m capable o ~detecting indirect as well as direct acting chemical carehaogens. Williams report~,~d the induction o f UDS in on'[y 74% o f the cells trea~],ed wiLth AAF [11]. Prelim.~aary results m this laboratory on the autoradmgraphic analys~s c,f AAF-mduced UDS svppofted these ~indmgs, therefore the repaY, capabilities of the unlabeled cells were questioned. The present ~ u d y was ~mdertaker,, L~ an a,~tempt to e x p h m this observa~,ion and to ~ur~her mwestiga~e t~e inducl~lon o f UDS m the p~imary rat hepatocyte culture ~ystem. MATI~RIA]~.SAND METHODS Hepatocytes were isolated from 3,-month-old Sprague--I)awley rats by the nl situ prefusmn o f the fiver using modifications incorporated from several techniques [1,3,8]. After isolatJLon, ceils were suspended in Ham's F-12 r.aedium (Microbmlogical Associates, Bethesda, Maryland) containing 17% fetal bov~ae serum (Grand Island BicAogical Company, Grand Island, New York) (GIBCO), 90.0 ~g/ml streptomycin, and 10.0 mU/ml insulin (8ign,~a Chemical Company, St. Louis, Missouri), referred to as the plating mediuxn throughout the remainder of this study. Vmbihty was asses~,~edby trypal~ blue e~elusion and the ee.'Is plated in 60-ram tissue-culture dishes at a deasltF o f 1.5 X 106 ~nable cells/plate and incubated at 37°C in an atmosphere of ~1~5% air and 5% CO2. After a 1--2 h period for the attachmenL o f viable cells,, ~,he plates were washed ,~ith Hank's balanced salt solution (HBSS) (GIBCq:?) to remove non.wable cells. AAF (Aldrich Chemical Company, Milwaukee, Wisconsm), 98.3% p~l'e by gas chromat.ography, and N-OH AAF, prepared as previously described [I4] and supplied by C. Weeks (National Center for Toxicolotpcal Research, Jefferson, Arkansm), were dissoNed in dimethyl sul~oxlde (DMSO). Appropnate quantitins o f each chemical were then added to plating medium containing I[0.0 gCi/ml [methyl-3H]thymldme (.40--60 Cl/mmol, New England Nv ear, Boston, Massachusetts) and unmedmtely added to the attached wabm eells. Ultrawo~et (UV) hght irradiatiort, ~[one and m conjanetion with chermea/ treatment, was used as a positive control, eliminating cytotoxmity as a possible e~lplanation for unindueed UDS [7]. All flmds were removed prior ~o irradmt~on of cells with UV light, and plating medium containing [3I-I[]thyre,dine was addefl immediately after irradia~tmn. All plates were mcubated ~br 24 h with AAF and N-OH AAF present throughout tbp entire period Rats treated wxLb 3-MC (Sigma) were rejected iatraperitoneally with 3-MC (100 mg/kg) 24 h before hepatocyte isolahon. For 5he a~ttoradingraphJc analysis of incorporated [ 3HI thymldme, the ccqls wet= l~xed in men,hanoi : ace~e acid (3 : 1), coated with a 5 : [ flflatmn of
175 N T B - 2 e m u l s i o n ( E a ~ m a r , !Kodak, R o c h e s t e r , N e w Y o r k ) , a n d d e v e l o p e d ,~fter a 14-day exposure period. Nuclear gn~ns fi'om 50 chosen at r e ~ d o m were c o u n t e d a n d t h e n u c l e a r a~eas m e a s u r e d . T h e s e c o u n t s w e r e ~,hen n o r m a h z e d ~o a n a r e a o f 1 0 0 / ~ m ~ a n d c o n ' e c t e d £br cyt~)plasmm b a c k g n r o u n d y m l d m g t h e c o r r e c t e d n u c l e a r grmns/10O ,urn ~. RESUL'~PS A N D ]D~rSCUSSION The incorporation of ['~H]~hymMme induced by 24-h incubation with A A F :,uad N - O H A A F , q u a n t l t a t e d b y r a d i o a c t i v i t y d e t e r m i n a t i o n s o n m a c r o m d e cules f r o m cell ]ysates, is s h o w n m Fig. 1A. A d o s e - d e p e n d e n t increase m t L e AAF ( . O / , d )
i
/ r
i ~]
.....................
......
ii--
// i~
:,,_ ........
!
,//
o i~,~
o 25J)
o 5oo N-OH A~r (.o/ml)
z oe~
Fig. i Quan(ltatlve i~alysls of AAF- and N-OH A~ F-reduced UDS by radmactzwty determmatmn on maeror~Loleeulas m cell lysates Cells were isolated and treated as described in Materials and Methods The cells were then harvested and eent,rffused to a pellet before lysmg in a 0.5% sodium dodeeyl sulfate ,,olutmn Ahquots wel e then made 5 % tn,ehloroace tie acid arid the plee~pltated macromolecumscollected on glas.; fiber 1flters for ~hc determination of [3H]th~ ra:dme incorporated by sclnttllation '~;pectmmetry The ]:)NA content of the filters was then detenained by a raod]tLc~tlon of ~he d~hera¢lamme method of Burton ['2]. The contrc4 (~) was incubated w~th ra~,dmra containing [~H]thym~d~ne and ().~% DMSO. ~he mean and the standard error of the ' nean are expressedl for each treatme,*ltt group (A) Hepatocytes ~solated from en untreated rat (~3) Hepatocy~es ~solated t¥om a rat m~ected mtrapentoneally w~th 10t) rag/kg L-MC 24 h pnor to ce]ll Isolation
176
specific activity of the D N A was seen after treatment wi~h N - O H A A F . All doses of A A F increased levels of [3H] thymidine incorporr~tion above that of the control, but there was no dose-c[ependent increase in t:hisincreased UI)S. A u t o m d i o g a p h i c analysis (Table 1) supports these findini~s,a~'well as demonstrating a m a ~ : i m u m of only 6 5 % of lhe cells induced to repair their I)NA. The col~abmed .A_AF and U V light treatment demonstrates that at least 8 7 % of the cells had the capability of repaying thear D N A . The N-hydroxylation of A A F is requzred for iCs metabolic conversion to N - O H A A F [[9] before, being fresher metabolized by sulfotransferase or o~her en:,wme systems [6] to an electrophilic ester capable of in!leracl;ingwith D N A . Lotlikar et al. [5] demonstrated that the conversion of A A F to N - O H A A F by
TABLE 1 A U T O R A D I O G R A P H I C SaNALYSIS OF T H E E F F E C T OF 3-MC T R E A T M E N T ON AAF'- A N D N-OH A A F - I N D U C E D UDS IN PRI~ClARY R A T HEPATOCYTEII~ Trea ~ment
Control
No 3-MC
3-MC Tr e a ted a
Corrected % Repamng c n acleargramsb /
Corrected % Repmr~ngd n uelear grainsb /
100 ~m 2
1O0 ~m:
2.8L1 ± 0 . 9 3
0 93 ± 1.22
2 2 2 c'rg/mm ~ UV hght
1'7.71 ± 1.72
79
~2 41 ± 5 . 4 4
92
N-OII A A F (ug/ml) 0.125 0.500 1 000
17.51 ± 2.15 31.32 ± 4.40 30 07 ± 3 . 5 5
3 3 ( 1 )e 85 79(2)
9.32 ± 1.78 2 2 . 0 6 ~ 1,96 55.~8 ± 4 . 7 6
25(5)0 75(6) 98
2 4 . 4 3 ± 2.02
81
57 7 2 ± 5 9 4
93
19 0 6 ± 2.75 19 50 ± 2 27 1 7 . 0 4 ± 2.40
65 56 55(3)
19.37 ± 2.54 39.27 ± 5 37 49 69 ~ 5 14
23 29 ± 2.23
87(4)
7 2 . 7 6 ± 7.39
0.5 ~g/ml N-OH A A F +222 erg/mm" UV hght A A F (~g/ml) i 25 b 00 10 O0 5.0 ~g/ml A A F +222 e r g / m m 2 UV hght
50(7) 8G 10'~(3) 89
a I00 mr, 'ks 3-~¢IC(in tnoctanom). IbNumbe~ of grams ± the 95% .confidencehtmlts. ~'Cellsshowmg U D S (;~ 13corrected grams/nucleus,P < 0.04 assuming Polssou with ,co~trolmean = (2.34 + 0.93 ). d Cellsshowing U D S (;~ 5 corrected gralns,/nucleus,P < O ~)6assuming Polsson with control mean = (0.93 + 1.22) e 2 ;~ 1 , 4 ;~ 3 , 6 ;* 5 , 8 ~ 7 and 8,~ 3 , P ~
0 01 (Chl-square test)
177 rat liver homogena~.~s was detectable only a~ter t r e a t m e n t o f t h e rat ~zd~h 3-MC prior to t h e h o m o g e n a t e preparatmn. 'l'he effect o f 3o]/~[C treatmen~ 24 h prior' ~,c, hepatocy~e isolation on t h e ind,~ction of UDS by A A F and N-OH A A F ~,ss h o w n in F~:g. l B . Both N-OH and A A F induced a dose-dependent J~crease in IYDS. The corrected nuclear grain c o u n t s f r o m t h e autoradiographic analysis agab~ support these findings (Table 1). in addition to t h e apparen~s increase in UDS/cell, t h e i:*ezcentage or cells induced to repair their DNA increased a~, t h e hig[~er dose level~.. These results suggest t h a t hepatic ce]~s isolated from uninduced animals retain suJ'fmient e n z y m a t i c capacity to metabolically activate N-OH AAF. These, results also ~mgges,t t h a t t h e cells' N-hydroxylating enzymes are present at ~low levels or are unstable in primary culture. q['he pze,,ent s t u d y confiz~ns t h e observat~on t h a t priming, rat hepa~ocyte cultu~es in conjunction w:[1;h UDS can ~,~erve as a valuable system to de~;ect potenll,ial cazcinogens i[10,~ 1]. The present, s t u d y also suggests that the sensiti ~it:~ o f this system m a y be enhanced, at least [or a r c m a t m amme~ and perhaps fol other chel~tlCal classes~ by 3-MC, t r e a t m e n t prior to nepatocyte isolation..4dditional chemical classes a~e n o w being tested to confine; th~s hypothesis. ACKNO
WLED(.~EMENTS
Thls ~ork ~s in partial fulfilmen~ by J.W. Oldham o f t~e r e q u ~ e m e n t s for t h e de$'ee of Doctor o£ Phdosophy m Intc~disciplinary Toxicology in the Gr~luate College, The University of Arkansas for Medmai Sciences and is s~pport,~d in part by U.S.P.H.S. Grant No. GM 21446. REFERE~CES
1 BDnn~y, R,J (1974) Adul~ hver parenehymal cells m primary culture Character~stic~ ~nd ceU rec~ogmtlo~Lstandards. In Vitro, 10,130--142 2 Burtor, K. (1968) Det~rrn/natlon of DNA concentration with dlphenylamme In Methods m Enzyrnology, ~oL 12B, pp 163--16~3 Editors L Giossman and K Mol[da~e. A,;ademie Press, ~4"ewYork 3 Latshes, B.~k. and Wdllams, G.M. (1976j Condltlons affecting prlmary cell cultures of t'unct]o~d ~dult ra#,hepatocytes. L The effect ol msuhn In Vitro, 12, 521--532 4 Lthk~',P.]).,Mdler, E.C, ~/hller,J,A, ~Id Margreth, A. ~19,p6} The enzymatLc reductlon oJ~ the ~V-hydr~,pxyder1~atives of 2-acetylammofluorene ~:! related carcinogens by tissue;prq.paratmns, Cancer Res., 25, 17-13~1762. 5 L tlikar P ]).,:Enomoto,M .Miller,J.A and Mdlcr. E.C (1967], Spemes val~atlon in the, N- and rlng-hydrox~latlon of 2-acel,ylammofluorene and effg~, of 3-metbtyLcholan1,hl~ene.~roc, Soc. Ezp. Biol ivied, ~L25,341--3,~,~. 6 Mtflder, q}.J, Hmsolft, J.A., Nelson, W.L and Thc rgeir~sson,S ,q.(1977) Role of sulfo-
trans[era:e from rat hver in tile mntagenlclty of N hydroxy-2 lcetTlaminoflt~orene m Salrnonel'a ~vph~mur,~m. B~ochem PharrnacoL, 2'~, 1356--1~ 58 7 Oldhl~, J.~., Casciano, D.A and Cave, M D (19'77) Dose-response mduet~on of DNA ~epa~r by =llrawolet hght in primary rat hepa~ocyt,~s J. Cell 1 ml, 75, 83a g Wagle,S }~. and ~ngebretsen, W.R (]975) Isolation, purification, and meLabohc
178 charactenshcs of rat liver hepatocytes. In. Methods m Enzymolvgy, VoL 35, pp: 579--594. Editor: J.M. Lowenstein, Academic Press, New York. 9 Weisburgcr, J.H. and We~bl=rger, JE.K. {1973) Biochemic~ Jornoa'~lon and pharmacological. ~cx~coiog~ca~ and pathological pro per~ms of hydroxylam,~es and hydroxamic acids. Pharmacol. Rev., 25, 1--66. 10 Wllliar~s, G.M. (1976) Carclnogen.indueed DN A repalr ln'p:~mazy rat bver ee~.l culturcs" a p osslbh screen for chemicel carcinogens. Cancer Letters, ]1, 251--236. i i WllI.:a~% ~ .M (] g77~ Detee~Jo~ c,f o h , ~ ; e ~ carc,f,~gens Dy unscheduled DNA synthesm m rat hw.~rpnmary cell cultures. Cancer Res. 37, 1¢~45--1,851.
173
2.ACETYLAM~b~OFLUOEE} ~E-~NDUC~D DNSC~',DULED ])I~A SYNTHE,%~S L'~.~ P A T O C Y T ~ , S I S O L A T E D FI~OM 3 . M F . T H Y L C H O L A b r r H ~ E N E TREA'J?ED I~ATS ]).A.CASCIANO and J.A.F A R R Department o f Health, ]3'd~eat~on c,n d Welfore, ]~bod and Drug Adm~ni~tra~ion, *Nati~)md Center for Toxicolo~cal R~seareh, Jefferson, Arhan~as 72079 (U.S.A.)
J.W. OLDHAM[a~d M.D. CAVE The Unfwersity of Arhanaas for Medical ~,c.~ences, Graduate Program for il~terdisciplinary 'Fox~eology, D~,partmen¢ o f Anatomy 456'1 W, Marh~am, Little Roch, Arhcnsas 7220~ IU, S.A )
(Received 11 hd[ay1973) (AeeepCed 2 June 1978)
The dose-dependent induction of unsehedu]ed DNA synthesis (lJI~S) by 2.acety']~ninofluorene (AAF) and N-OH-2-ace~y}amino:{]uerene (N.GI'I AAF) ~,~primary rat hepatocytes iso,Latedfrom un~'eated and 3-methyJcholantlu'e.lle (3-MC) treated rats was invesl,lgated. 3-MC treatment w~as not necessary for ~he dose-dependent induction of U D S fmduced by N - O H A A F , sug~e:~tingthe presence of enzyme levels adequate for its estenfication to ~m active form in il;olated primary hepatocy~s. Although all doses of AAF increased the level of [SH][thymid£ae incc,rporationabove tha~ of ~he control, a dose-dependenl~ response could not be demonstrated, suggesting inadequate constitutivelevels of N-hydroxyla[,~ng enzymes. Treatment of rats with 3-MC 24 h prier to hepatocyte isolation:~sulted ia a dose-dependent induction of U D S b'.¢AAF. ..MC treatment increased the amount of U D S per cell,as wel~ as the percentage ef cellsinduced to repairtheir D N A .
IN T RO]'},UCTION
The use of the primary ra~ hepatoq~,te cuRure in col~junetmn wi~';hthe Address correspondence to D.~. Casclano, ~n.D , DlL~lmonof Mutagenesls Research, I-IF'~120, Natlona! Center for Toxicological Research, Jefferson, Arkansas 7207~.), qJ.SA Ab~.rev~itionJ~. AAF, 2-ace~ylmI~~ofluorene;DMSO, dhrne~yl su~foxltde;HBSSo Bank's
ba]ta.acedsaltsolution;~-It~C,.bmethylcholanthrene,N-OH AFF, N-OH-2-aeetylaminoflaol,'ene;UDS, um~,cheduledD N A synthesls;UV, ultraviolet
174 induction of UD,S as a predictive system for potential carcinogenic activity o f a ehemk:al compound was first suggested by Williams [10,11]. The hl,epatocyte not~ only serves as the target for DNA dmnage and its subsequent repair by a given chemical, but also contains constitutive levels of enzymes necessm'y for activa~,ion o f p~ecareinogens to ~heir reactive form. The system is therefo~m capable o ~detecting indirect as well as direct acting chemical carehaogens. Williams report~,~d the induction o f UDS in on'[y 74% o f the cells trea~],ed wiLth AAF [11]. Prelim.~aary results m this laboratory on the autoradmgraphic analys~s c,f AAF-mduced UDS svppofted these ~indmgs, therefore the repaY, capabilities of the unlabeled cells were questioned. The present ~ u d y was ~mdertaker,, L~ an a,~tempt to e x p h m this observa~,ion and to ~ur~her mwestiga~e t~e inducl~lon o f UDS m the p~imary rat hepatocyte culture ~ystem. MATI~RIA]~.SAND METHODS Hepatocytes were isolated from 3,-month-old Sprague--I)awley rats by the nl situ prefusmn o f the fiver using modifications incorporated from several techniques [1,3,8]. After isolatJLon, ceils were suspended in Ham's F-12 r.aedium (Microbmlogical Associates, Bethesda, Maryland) containing 17% fetal bov~ae serum (Grand Island BicAogical Company, Grand Island, New York) (GIBCO), 90.0 ~g/ml streptomycin, and 10.0 mU/ml insulin (8ign,~a Chemical Company, St. Louis, Missouri), referred to as the plating mediuxn throughout the remainder of this study. Vmbihty was asses~,~edby trypal~ blue e~elusion and the ee.'Is plated in 60-ram tissue-culture dishes at a deasltF o f 1.5 X 106 ~nable cells/plate and incubated at 37°C in an atmosphere of ~1~5% air and 5% CO2. After a 1--2 h period for the attachmenL o f viable cells,, ~,he plates were washed ,~ith Hank's balanced salt solution (HBSS) (GIBCq:?) to remove non.wable cells. AAF (Aldrich Chemical Company, Milwaukee, Wisconsm), 98.3% p~l'e by gas chromat.ography, and N-OH AAF, prepared as previously described [I4] and supplied by C. Weeks (National Center for Toxicolotpcal Research, Jefferson, Arkansm), were dissoNed in dimethyl sul~oxlde (DMSO). Appropnate quantitins o f each chemical were then added to plating medium containing I[0.0 gCi/ml [methyl-3H]thymldme (.40--60 Cl/mmol, New England Nv ear, Boston, Massachusetts) and unmedmtely added to the attached wabm eells. Ultrawo~et (UV) hght irradiatiort, ~[one and m conjanetion with chermea/ treatment, was used as a positive control, eliminating cytotoxmity as a possible e~lplanation for unindueed UDS [7]. All flmds were removed prior ~o irradmt~on of cells with UV light, and plating medium containing [3I-I[]thyre,dine was addefl immediately after irradia~tmn. All plates were mcubated ~br 24 h with AAF and N-OH AAF present throughout tbp entire period Rats treated wxLb 3-MC (Sigma) were rejected iatraperitoneally with 3-MC (100 mg/kg) 24 h before hepatocyte isolahon. For 5he a~ttoradingraphJc analysis of incorporated [ 3HI thymldme, the ccqls wet= l~xed in men,hanoi : ace~e acid (3 : 1), coated with a 5 : [ flflatmn of
175 N T B - 2 e m u l s i o n ( E a ~ m a r , !Kodak, R o c h e s t e r , N e w Y o r k ) , a n d d e v e l o p e d ,~fter a 14-day exposure period. Nuclear gn~ns fi'om 50 chosen at r e ~ d o m were c o u n t e d a n d t h e n u c l e a r a~eas m e a s u r e d . T h e s e c o u n t s w e r e ~,hen n o r m a h z e d ~o a n a r e a o f 1 0 0 / ~ m ~ a n d c o n ' e c t e d £br cyt~)plasmm b a c k g n r o u n d y m l d m g t h e c o r r e c t e d n u c l e a r grmns/10O ,urn ~. RESUL'~PS A N D ]D~rSCUSSION The incorporation of ['~H]~hymMme induced by 24-h incubation with A A F :,uad N - O H A A F , q u a n t l t a t e d b y r a d i o a c t i v i t y d e t e r m i n a t i o n s o n m a c r o m d e cules f r o m cell ]ysates, is s h o w n m Fig. 1A. A d o s e - d e p e n d e n t increase m t L e AAF ( . O / , d )
i
/ r
i ~]
.....................
......
ii--
// i~
:,,_ ........
!
,//
o i~,~
o 25J)
o 5oo N-OH A~r (.o/ml)
z oe~
Fig. i Quan(ltatlve i~alysls of AAF- and N-OH A~ F-reduced UDS by radmactzwty determmatmn on maeror~Loleeulas m cell lysates Cells were isolated and treated as described in Materials and Methods The cells were then harvested and eent,rffused to a pellet before lysmg in a 0.5% sodium dodeeyl sulfate ,,olutmn Ahquots wel e then made 5 % tn,ehloroace tie acid arid the plee~pltated macromolecumscollected on glas.; fiber 1flters for ~hc determination of [3H]th~ ra:dme incorporated by sclnttllation '~;pectmmetry The ]:)NA content of the filters was then detenained by a raod]tLc~tlon of ~he d~hera¢lamme method of Burton ['2]. The contrc4 (~) was incubated w~th ra~,dmra containing [~H]thym~d~ne and ().~% DMSO. ~he mean and the standard error of the ' nean are expressedl for each treatme,*ltt group (A) Hepatocytes ~solated from en untreated rat (~3) Hepatocy~es ~solated t¥om a rat m~ected mtrapentoneally w~th 10t) rag/kg L-MC 24 h pnor to ce]ll Isolation
176
specific activity of the D N A was seen after treatment wi~h N - O H A A F . All doses of A A F increased levels of [3H] thymidine incorporr~tion above that of the control, but there was no dose-c[ependent increase in t:hisincreased UI)S. A u t o m d i o g a p h i c analysis (Table 1) supports these findini~s,a~'well as demonstrating a m a ~ : i m u m of only 6 5 % of lhe cells induced to repair their I)NA. The col~abmed .A_AF and U V light treatment demonstrates that at least 8 7 % of the cells had the capability of repaying thear D N A . The N-hydroxylation of A A F is requzred for iCs metabolic conversion to N - O H A A F [[9] before, being fresher metabolized by sulfotransferase or o~her en:,wme systems [6] to an electrophilic ester capable of in!leracl;ingwith D N A . Lotlikar et al. [5] demonstrated that the conversion of A A F to N - O H A A F by
TABLE 1 A U T O R A D I O G R A P H I C SaNALYSIS OF T H E E F F E C T OF 3-MC T R E A T M E N T ON AAF'- A N D N-OH A A F - I N D U C E D UDS IN PRI~ClARY R A T HEPATOCYTEII~ Trea ~ment
Control
No 3-MC
3-MC Tr e a ted a
Corrected % Repamng c n acleargramsb /
Corrected % Repmr~ngd n uelear grainsb /
100 ~m 2
1O0 ~m:
2.8L1 ± 0 . 9 3
0 93 ± 1.22
2 2 2 c'rg/mm ~ UV hght
1'7.71 ± 1.72
79
~2 41 ± 5 . 4 4
92
N-OII A A F (ug/ml) 0.125 0.500 1 000
17.51 ± 2.15 31.32 ± 4.40 30 07 ± 3 . 5 5
3 3 ( 1 )e 85 79(2)
9.32 ± 1.78 2 2 . 0 6 ~ 1,96 55.~8 ± 4 . 7 6
25(5)0 75(6) 98
2 4 . 4 3 ± 2.02
81
57 7 2 ± 5 9 4
93
19 0 6 ± 2.75 19 50 ± 2 27 1 7 . 0 4 ± 2.40
65 56 55(3)
19.37 ± 2.54 39.27 ± 5 37 49 69 ~ 5 14
23 29 ± 2.23
87(4)
7 2 . 7 6 ± 7.39
0.5 ~g/ml N-OH A A F +222 erg/mm" UV hght A A F (~g/ml) i 25 b 00 10 O0 5.0 ~g/ml A A F +222 e r g / m m 2 UV hght
50(7) 8G 10'~(3) 89
a I00 mr, 'ks 3-~¢IC(in tnoctanom). IbNumbe~ of grams ± the 95% .confidencehtmlts. ~'Cellsshowmg U D S (;~ 13corrected grams/nucleus,P < 0.04 assuming Polssou with ,co~trolmean = (2.34 + 0.93 ). d Cellsshowing U D S (;~ 5 corrected gralns,/nucleus,P < O ~)6assuming Polsson with control mean = (0.93 + 1.22) e 2 ;~ 1 , 4 ;~ 3 , 6 ;* 5 , 8 ~ 7 and 8,~ 3 , P ~
0 01 (Chl-square test)
177 rat liver homogena~.~s was detectable only a~ter t r e a t m e n t o f t h e rat ~zd~h 3-MC prior to t h e h o m o g e n a t e preparatmn. 'l'he effect o f 3o]/~[C treatmen~ 24 h prior' ~,c, hepatocy~e isolation on t h e ind,~ction of UDS by A A F and N-OH A A F ~,ss h o w n in F~:g. l B . Both N-OH and A A F induced a dose-dependent J~crease in IYDS. The corrected nuclear grain c o u n t s f r o m t h e autoradiographic analysis agab~ support these findings (Table 1). in addition to t h e apparen~s increase in UDS/cell, t h e i:*ezcentage or cells induced to repair their DNA increased a~, t h e hig[~er dose level~.. These results suggest t h a t hepatic ce]~s isolated from uninduced animals retain suJ'fmient e n z y m a t i c capacity to metabolically activate N-OH AAF. These, results also ~mgges,t t h a t t h e cells' N-hydroxylating enzymes are present at ~low levels or are unstable in primary culture. q['he pze,,ent s t u d y confiz~ns t h e observat~on t h a t priming, rat hepa~ocyte cultu~es in conjunction w:[1;h UDS can ~,~erve as a valuable system to de~;ect potenll,ial cazcinogens i[10,~ 1]. The present, s t u d y also suggests that the sensiti ~it:~ o f this system m a y be enhanced, at least [or a r c m a t m amme~ and perhaps fol other chel~tlCal classes~ by 3-MC, t r e a t m e n t prior to nepatocyte isolation..4dditional chemical classes a~e n o w being tested to confine; th~s hypothesis. ACKNO
WLED(.~EMENTS
Thls ~ork ~s in partial fulfilmen~ by J.W. Oldham o f t~e r e q u ~ e m e n t s for t h e de$'ee of Doctor o£ Phdosophy m Intc~disciplinary Toxicology in the Gr~luate College, The University of Arkansas for Medmai Sciences and is s~pport,~d in part by U.S.P.H.S. Grant No. GM 21446. REFERE~CES
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