The effects of CCl4 on the content of nicotinamide adenine dinucleotide phosphate in rat liver

Chem.-Biol. Interactions, 16 (1977) 359--364 © Elsevier/North-Holland Scientific Publishers, Ltd.

359

Short Communication THE E F F E C T S OF CC14 ON THE CONTENT OF NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE IN R A T L I V E R

T.F. SLATER and B.C. SAWYER Department of Biochemistry, Brunel University, Uxbridge, Middlesex (Great Britain)

(Received December 10th, 1976) (Accepted December 16th, 1976)

The hepatotoxic action of CCL in the rat is known to be dependent on a metabolic activation of the toxic agent to a highly reactive p r o d u c t probably the trichloromethyl radical (for background references see refs. 3 and 5). The activation occurs through an interaction with the N A D P H - c y t o c h r o m e P-450 electron transport chain that is associated with the endoplasmic reticulum. The production of a highly reactive moiety such as CCI~ within the lipid environment of the endoplasmic reticulum results in the initiation o f lipid peroxidation, to rapid disturbances to neighbouring enzymes such as cytochrome P-450 and glucose-6-phosphatase, and to covalent binding of CCI~ to lipid and protein (see ref. 5). Although the precise locus (or l o c i ) o f activation of CC14 along the NADPH-cytochrome P-450 chain is still controversial (for example see ref. 2) many data are consistent with one locus being near to the NADPH-flavoprotein [9]. Such an activation site would present favorable conditions for an interaction of the activated product (CCI~) with the reducing source (NADPH). Although a decrease in NADPH is k n o w n to be one of the early consequences of CC14 intoxication [6] neither the dependence of this effect on the dose of CC14 administered, nor the long-term time dependency of the effect produced have been reported; these data are reported here. Materials and m e t h o d s

Female albino rats, b o d y wt. approx. 150 g were used; t h e y were fed M.R.C. diet 41B and water ad libitum until 18 h before administration of CC14 when f o o d was removed. CC14 was diluted with liquid paraffin (the concentrations used are given in the text) and was administered b y stomach tube (0.5 ml solution/100 g b o d y wt.) with the rat under light ether anaesthesia; control rats received an equivalent volume of liquid paraffin. The rats were killed b y cervical dislocation at various times after dosing, and pieces of liver were immediately removed for nucleotide assay using the m e t h o d of Slater et al. [ 7 ].

360 Results The effects of various concentrations of CC14 on NADP ÷ and NADPH in liver 24 h after dosing are shown in Table I. It can be seen t h a t the major change produced was in the content of NADPH; a significant decrease being observed even with a very low dose of CC14 (51 pmoles/100 g b o d y wt.). This change in NADPH was dose-dependent; the percentage decreases in NADPH (pg/liver/100 g b o d y wt.) with increasing doses of CC14 as pmoles/ kg b o d y wt. in parentheses were: 16% (34 pmoles CC14) 14% (51 pmoles CC14) 33% (103 #moles CC14), 35% (258 pmoles CC14), 40% (1290 pmoles CC14), 51% (2580 pmoles CC14). The effects of 258 pmoles CC14/100 g b o d y wt. on liver NADP ÷ and NADPH at various times after dosing are shown in Table II. It can be seen that the m a x i m u m depression occurred 24--48 h after dosing; afterwards there was a slow return to normal values. Discussion The results shown in Table I illustrate the sensitivity of liver NADPH to the presence of CC14. Previous data on the changes in NADPH content after CC14 administration [6] were mainly concerned with the dose level of 1290 pmoles/100 g b o d y wt.; the present results show that similar decreases in liver NADPH content can be achieved with 100 #moles/100 g b o d y wt. Associated with the decrease in NADPH is a change in the NADPH/NADP ÷ ratio in favour of a more oxidised condition. The decrease in NADPH content observed here is consistent with previous suggestions [9] that CC14 is activated to the trichloromethyl radical at or near to the NADPH-flavoprotein. The production of CCI~ at such a locus can be expected to result in loss of NADPH since it is k n o w n [8] that CCI~ interacts rapidly with reduced nicotinamide adenine dinucleotides with consequent loss of coenzyme activity. The decrease in whole liver NADPH content observed with the dose range 100--1250 pmoles/100 g b o d y wt. was approx. 30% (Table I). It is usually assumed, and generally accepted as a working assumption, t h a t CC14 is activated to a toxic product only in the parenchymal cells of the centrilobular region, thereby accounting for the centrilobular localisation of the liver necrosis produced by CC14 (see refs. 3--5). If this assumption is true, then it can be speculated that the decrease in NADPH observed in whole liver samples (as measured here) represents a much bigger decrease in the NADPH c o n t e n t of centrilobular cells, with a smaller decrease in cells of the mid-zone and periportal zones. If, indeed, this is the situation in vivo, then a major result of CC14 intoxication may be a severe or even total depletion of NADPH in the centrilobular zone; this can be expected to produce profound metabolic disturbances in the affected cells t h a t eventually is manifest as centrilobular necrosis. The higher initial concentrations of NADPH in the liver cells achieved by pre-dosing with nicotinamide [1] may prevent NADPH from being depleted below a critical level following CC14 administration, and this could be of relevance to the reported protective effects of nicotinamide in this type of intoxication.

22 -+ 2(5) 26 -+ 4 ( 6 ) a

27 + 4 ( 6 ) 32 -+ 3(6) a

27 -+ 1(4) 33 -+ 1(8) b

26 -+ 1 ( 2 6 )

Control 258 pmoles/ 100 g body wt.

Control 1290 pmoles/ 100 g body wt.

Control 2580 pmoles/ 100 g b o d y wt.

All c o n t r o l s 0.05; d p<

27 -+ 4 ( 3 ) 33 + 2(7) a

Control 103 p m o l e s / 100 g body wt.

0.1;c p~

24 + 1(4) 27 + 2(4) a

Control 52 p m o l e s / 100 g b o d y wt.

a NS; b p ~

30 -+ 6 ( 4 ) 34-+1(4)a

0.01;e p<

135 + 8 ( 2 6 )

125 + 18(4) 164 + 7(8) e

129 + 14(6) 1 8 0 -+ 2 1 ( 6 ) b

107 + 1 2 ( 5 ) 134 + 1 8 ( 6 ) a

145 + 2 7 ( 3 ) 155 + 1 0 ( 7 ) a

122 + 10(4) i38 + 19(4) a

162 + 3 0 ( 4 ) 182+ 7(4)a

0.001.

2 1 9 -+ 6 ( 2 6 )

2 4 9 -+ 1 6 ( 4 ) 1 0 4 -+ 1 1 ( 8 ) e

222 + 15(6) 1 2 2 -+ 13(6} e

207 + 10(5) 131 + 9 ( 6 ) e

2 0 8 -+ 6(3) 154 -+ 1 5 ( 7 ) b

1 9 2 -+ 1 4 ( 4 ) 169 + 14(4) a

233 + 19(4) 202+ 9(4)a

pg/g

pg/g

/Jg/liver/ 100 g body wt.

NADPH

NADP ÷

Control 34pmoles/ 100 g b o d y wt.

Dose o f CC14

1 0 8 4 -+ 3 4 ( 2 6 )

1 0 9 7 -+ 5 5 ( 4 ) 537 -+ 6 8 ( 8 ) e

1 0 9 9 -+ 7 9 ( 6 ) . 6 6 3 -+ 7 3 ( 6 ) a

1032 + 96(5) . 6 7 6 -+ 3 6 ( 6 ) d

1 0 4 0 -+ 9 2 ( 3 ) . 702 + 31(7) d

9 8 2 -+ 2 8 ( 4 ) 849 + 36(4) e

1 2 5 3 -+ 8 7 ( 4 ) 1057+75(4) a

pg/liver/ 1 0 0 g b o d y wt.

245 +

7(26)

2 7 6 -+ 1 5 ( 4 ) 137 -+ 1 0 ( 8 ) e

249 + 17(6) . 154 + 1 3 ( 6 ) d

229 -+ 1 1 ( 5 ) 159 + 1 1 ( 6 ) d

235 + 6(3) 187 +-- 1 7 ( 7 ) a

216 +- 1 4 ( 4 ) 196 -+ 1 2 ( 4 ) a

268 -+ 2 0 ( 3 ) 236+ 9(4)a

pg/g

10.2+2.22(3) 5.8+0.37(4)

1400-+ 1239-+

9.8 + 0 . 7 2 ( 5 ) 5.4 + 0 . 7 4 ( 6 ) d

8.7 + 0 . 5 8 ( 6 ) 3.8 + 0 . 5 5 ( 6 ) e

9.1 + 0 . 9 4 ( 4 ) 3.4+0.55(8)e

1139 + 1 0 5 ( 5 ) 811 + 4 6 ( 6 ) c

1232 + 95(6) . 843 + 80(6) d

72(4) 63(8)e

36(26)

1223 + 701+

1212 +

0.38(24)

9.3 + 1 . 1 0 ( 2 ) 4.6 + 0 . 2 7 ( 7 ) e

1185 -+ 9 2 ( 3 ) 8 5 8 -+ 3 6 ( 7 ) d

9.2

8.2 + 0 . 7 8 ( 4 ) 6.6 -+ 1 . 0 5 ( 4 ) a

b

1 1 0 5 -+ 2 7 ( 4 ) 987 + 18(4) c

67(4) 78(4)a

NADP+

NADPH

pg/liver/ 100 g b o d y wt.

NADP ÷ and NADPH

Mean values + SEM are s h o w n , w i t h t h e n u m b e r s o f a n i m a l s used in e a c h e x p e r i m e n t given in p a r e n t h e s e s . T h e statistical a s s e s s m e n t o f differences b e t w e e n m e a n values was p e r f o r m e d using S t u d e n t ' s t-test.

E F F E C T S O F V A R I O U S D O S E S O F CC14 ON T H E N A D P H A N D N A D P ÷ C O N T E N T S O F R A T L I V E R S A M P L E S

TABLE I

II

258 ~MOLES

CC14/100

a Ns;b

p<

All c o n t r o l s

0.1;e

p<

Control CC14

Control CCI4

72h

12 days

Control CC14

48h

Control CCI 4

Control CC14

24 h

5 days

Control CC14

Treatment

3 h

Time

0.05;d

p<

25 + 2(33) p<

122 ± 0.001.

7(32)

128 ± 16(9) 1 1 7 + - 8( 9) a

127 ± 10(4) 109± 4( 4) a

197 ±

5(33)

194 ± 10(10) 2 0 8 ± 1 4 ( 8) a

192 ± 15(4) 1 8 3 - + 7( 4) a

229-+ 15(4) 1 7 1 - + 4( 4) d

1 9 4 - + 1 1 ( 6) d 121-+ 19(6)

9 1 -+ 9 ( 6 ) 7 8 ± 1 1 ( 6) a 164 ± 28(4) 133± 9( 4) a

207 ± 10(5) 131± 9( 6) e

172 ± 14(4) 1 8 2 . ± 4( 4) a

107 ± 12(5) 1 3 4 - + 1 8 ( 6) a

1 2 9 +_ 2 5 ( 4 ) 1 2 6 ± 1 8 ( 4) a

0.01;e

27 ± 3 ( 1 0 ) 2 6 ± 2( 9) a

23 ± 1(4) 2 5 + 1( 4) a

32-+ 5 ( 4 ) 27± 3(4) a

-

20+ 1(6) a 17 + 3(6)

2 2 ± 2( 5)~a 2 6 + 4( 6 i

28 ± 6(4) 2 9 + 4( 4) a

~g/g

~g/liver] 100 g body wt.

pg/g

used shown

WT. ON THE

NADPH

of animals

G BODY

NADP ÷

Mean values are given + SEM with the number was performed using Student's t-test.

THE EFFECTS OF AFTER DOSING

TABLE

c

d

d

a

950 ±

31(32)

8 8 1 -+ 1 9 ( 9 ) 935± 5 1 ( 8) a

1064 ± 100(4) 809-+ 9( 4) c

1163 ± 79(4) 895± 39(4)

- 39(6) 869 + 554-+ 66(6)

1032 ± 96(5) 676± 36(6)

799 ± 54(4) 780± 9(4)

pgfliver/100 g body wt.

OF

assessment

d

2 2 3 -+ 6 ( 3 3 )

223 ± 13(10) 2 3 3 - + 1 5 ( 8) a

215 ± 15(4) 2 0 7 ± 8( 4) a

261 ± 20(4) 1 9 9 ± 4( 4) c

2 1 4 - + 1 0 ( 6) d 138± 21(6)

229 ± 11(5) 159 ± 11(6)

2 0 1 -+ 1 9 ( 4 ) 211± 6( 4) a

Dg/g

NADPH

of differences

NADP ÷ AND

44(6) 75(6)

36(28)

1008 ± 20(9) 1 0 4 7 +- 5 4 ( 8) a

1191 ± 102(4) 9 1 8 - + 1 1 ( 4) e

1093 ±

9.8 + 0.72(5) 5 . 4 -+ 0 . 7 4 ( 6 )

a

a

a

a

d

a

values

TIMES

8 . 6 +- 0 . 4 4 ( 3 1 )

8 . 2 -+ 0 . 7 8 ( 8 ) 8 . 6 -+ 0 . 7 4 ( 8 )

8.5 + 0.93(4) 7 . 4 -+ 0 . 2 7 ( 4 )

7 . 5 +- 0 . 8 4 ( 4 ) 6 . 5 -+ 0 . 8 4 ( 4 )

d 1 0 . 1 -+ 1 . 4 1 ( 6 ) 7.3 + 0.70(6) 1 3 2 7 -+ 1 0 7 ( 4 ) 9 7 8 - + 5 4 ( 4) c

960+ 632+-

1139 ± 105(5) 811± 46( 6) c

mean

6.8 + 1.04(4) 6 . 6 -+ 0 . 9 7 ( 4 )

NADP +

NADPH

between

AT VARIOUS

9 6 8 _+ 5 0 ( 4 ) 960± 18( 4) a

~g/liver/100 g body wt.

NADP + and NADPH

Statistical

CONTENT

in parentheses.

LIVER'S

b~

363 120 loo

~

"

~

|

8O z < uz

6O

'~ z

4o

x < E

2O

DAYS

Fig. 1 . Changes in liver cytochrome P-450 (e), NADPH-cytochrome c reductase (A), and NADPH (©) after dosing with CC14 (258 pmoles/100 g body wt.) at time zero. The values are shown as percentages of the maximum disturbances produced. The data for NADPHcytochrome c reductase and for cytochrome P-450 are taken from ref. 2.

The time course of the changes NADPH in the liver following CC14 administration are of interest to reports dealing with the site of activation of CC14 on the NADPH-cytochrome P-450 electron transport chain [2]. The changes produced in liver NADPH (this paper) in c y t o c h r o m e P-450 and in drug metabolising activity [2] following CC14 administration are shown in Fig. 1. It can be seen that the time courses of the changes are similar. In contrast, there is little change in the NADPH-cytochrome c reductase activity measured in microsomal suspensions [2] and this result was interpreted to indicate that CC14 was not activated at the flavoprotein site. However, such assays in vitro are routinely performed under optimal conditions (e.g. optimal NADPH) and yet, as the above discussion suggests, NADPH may itself become limiting in vivo in the affected cells of the centrilobular zone. In such circumstances, activation of CC14 at the flavoprotein locus m a y be restricted by the supply of NADPH whilst drug metabolism utilising the c y t o c h r o m e P-450 system may become limited b y both NADPH and P-450. Part of this work was carried o u t under generous financial assistance from CIBA-Geigy Ltd. to w h o m we are most grateful. 1 2 3 4 5 6 7 8 9

J.W. Gibb and T.M. Brody, Biochem. Pharmacol., 16 (1967) 2047. E.A. Glende, Biochem. Pharmacol., 21 (1972) 1697. R.O. Recknagel, Pharmacol. Revs., 19 (1967) 145. T.F. Slater, Nature (London), 209 (1966) 36. T.F. Slater, Free Radical Mechanisms in Tissue Injury, Pion, London, 1972. T.F. Slater, U.D. Striiuli and B.C. Sawyer, Biochem. J., 93 (1964) 260. T.F. Slater, B.C. Sawyer and U.D. Striiuli, Arch. Int. Physiol. Biochim., 72 (1964) 427. T.F. Slater and P.J. Jose, Biochem. J., 114 (1969) 7-8P. T.F. Slater and B.C. Sawyer, Biochem. J., 123 (1971) 815.