EXPEIUMENTAL AND MOLECULAR PATHOLOGY 21, 237-245 (1974)

Effect of Cycloheximide on the Distribution of a-Naphthylisothiocyanate in Rats

SIMON LOCK, HANSPETER WITSCHI,~~ 2 FREDIZFU~K S. &ELTON,

GEORGE HANASONO, AND GABRIEL L. PLAA 1

Dbpartement de pharmucologie, FacultS de mddecine, UniverstiS de Mont&al, Mont&al, Q&bee, Canada

Received March 15, 1974

Others have shown that in rats the administration of cycloheximide (2 mg/kg) within 2 hr before or after 300 mgjkg of or-naphthylisothiocyanate (ANIT) blocks the development of hyperbilirubinemia and cholestasis. The effects of cyclo- heximide on the tissue distribution of ANIT labeled with “C or ‘H were examined. In cycloheximide-treated animals, less ANIT-derived radioactivity was found in blood, liver, fat and several other tissues at various times after ANIT than in controls. Rats given a mixture of (naphthyl-4-‘H) ANIT and (isothiocyanate-*‘C) ANIT with a ratio of ‘H/%:6.3, excreted in their bile during the first 8 hr after ANIT, more “H compared to “C. In rats treated with cycloheximide, the ‘H/“C ratio

did not change. The data suggest that normal animals excrete an ANIT metabolite in their bile from which the “C label in the isothiocyanate moiety has been lost, whereas this does not happen in cycloheximide-treated animals. This provides further evidence that biotransformation of ANIT is necessary for the development of hyperbilirubinemia and cholestasis. Bile might be a convenient starting material for the isolation and identification of ANIT metabolites.

INTRODUCTION

cu-Naphthylisothiocyanate (ANIT) is a chemical that has interested patholo- gists and toxicologists for some time. Chronic administration of this substance causes bile ductule hyperplasia and biliary cirrhosis, while single oral doses produce both cholestasis and hyperbilirubinemia within 12-24 hr (Plaa, 1970). ANIT can therefore be used to produce and to study, in laboratory animals, hepatotoxic responses which are seen quite frequently in man as undesirable side effects to drugs.

We have recently reported that cycloheximide, 2 mg/kg given up to 24 hr before or up to 8 hr after a hepatotoxic dose of ANIT, modifies considerably the cholestatic and hyperbilirubinemic responses and that the animals can even be completely protected if the cycloheximide is given within 2 hr before or after the ANIT ( Indacochea-Redmond et al., 1973). Cycloheximide is best known for its almost instantaneous inhibition of protein synthesis in liver and other organs (Verbin et al., 1969; Verbin and Farber, 1967; Witschi 1973). How this can be related to the protective effect afforded against ANIT-in-

1 Member, MRC Group in Drug Toxicology. 2 To whom requests for reprints should be sent.

237

1974 by Academic Press,Inc. reproduction in any form reserwl.

238 LOCK ET AL.

p&S N-C-S

FIG. 1. Position of the radioactive Iabel in “C-ANIT (left) and in *H-ANIT fright).

duced hepatotoxicity remains an unsolved probIem. It has been shown by Capizzo and Roberts (1971b) that actinomycin D, which is capable of pro- tecting rats partially against ANIT ( Indacochea-Redmond et al., 1973) interferes possibly with absorption and biotransformation of ANIT. The experiments in the present study were designed to determine whether cycloheximide, which affords much better protection than does actinomycin D, would also affect absorption and biotransformation of ANIT.

MATERIALS AND METHODS

Male Sprague-Dawley rats, purchased from Bio-Breeding Laboratories, Ottawa, Ontario, and weighing between 160-230 g were used in all experiments. The animals were maintained on Purina Chow and water ad E&turn. ANIT was purchased from Eastman Kodak, Rochester, New York. Radioactive ANIT, labeled with 14C in the isothiocyanate moiety (Fig. 1) was purchased from International and Chemical Nuclear Corp. Irving, California; the specific activity was 1.05 mCi/mmole. ANIT labeled with 3H in the 4-position of the naphthalene ring (Fig. 1) was purchased from New England Nuclear, Waltham, Massachu- setts; the sp act was 259.3 mCi/mmole.

In Experiment I, cold ANIT and 14C-ANIT were mixed together and sus- pended with a homogenizer combining mechanical shear with ultrasound (Poly- tron tissue homogenizer, Brinkmann Instruments) in 1.5% carboxymethylcellulose sodium to give a final concentration of 30 mg/ml (1 &i/ml). The mixture was given to rats by gavage at a dose of 300 mg/kg, 10 &i/kg. Half of the animals had been pretreated 30 min before ANIT with an ip injection of cycloheximide (2 mg/kg); the other half received 0.9% NaCl ip and served as controls. At different times after ANIT administration, the animals were anesthetized with ether and a blood sample was withdrawn from the abdomina1 aorta, The entire liver, heart, both kidneys, and lungs were homogenized in ‘distilled water. Aliquots of the homogenates containing 100-200 mg of tissue were transferred into scintillation vials, mixed with 2 ml of tissue soIubilizer (Protosol, New England Nuclear), tightly capped and left overnight at 45°C. Aliquots of epididymal fat pad were directly digested with Protosol. After cooling, the samples were mixed with 10 ml of Aquasol (New EngIand Nuclear) and counted in a Packard Tri-Carb liquid scintillation spectrometer. Blood samples (0.2 ml) were spotted on circles of filter paper, air dried and burned in a Packard Tri- Carb sample oxidizer. Corrections for quenching were made with the help of automatic external standardization.

In Experiment 2, food was withdrawn from the animaIs the night before they were given I’C-ANIT, 300 mg/kg ( 10 &i/kg), Cycloheximide (2 mg/kg) was injected y2, 1, 2, 4, 8 or 24 hr before ANIT administration. Control animals

CYCLOHEXIMIDE AND ANIT DISTRIBUTION 239

received 0.9% NaCl ip at the same hours before ANIT. All animals were killed exactly 1 hr after ANIT and tissue samples were processed as described above.

Experiment 3 was identical to Experiment 1, except that 3H-ANIT (300 mg/kg, 50 &i/kg) was administered.

In Experiment 4, rats were lightly anesthetized with ether while the carotid artery and common bile duct were cannulated with polyethylene tubing. After recovery from anesthesia, experimental animals were injected with cycloheximide (2 mg/kg). Control animals received 0.9% NaCl ip. Thirty minutes later, all animals were given a mixture of cold ANIT (300 mg/kg) with ‘C-ANIT (20 &i/kg) and 3H-ANIT (100 &i/kg). Periodically, blood (0.2 ml) and bile samples (0.1 ml) were collected and spotted on filter paper discs; bile volume was measured and recorded. At the end of the experiment, various tissues were homogenized in distilled water. Aliquots of the homogenates were freeze dried and 10-100 mg of the dry powder was wrapped in filter paper and pressed into a pellet. The pellets and the filter papers containing blood or bile were burned in the sample oxidizer and the resulting 14C02 and 3H20 collected in separate vials; appropriate preliminary experiments had shown that separation of the two isotopes was complete. After stabilization, samples were counted and corrected for quenching by internal standardization.

PRESENTATION OF DATA

Although the chemical compound(s) associated with the radioactivity mea- sured in blood and the different tissues after administration of 14C-ANIT or 3H-ANIT were not identified, we corrected all dpm into pg of ANIT equivalents. This was done in order to facilitate a direct comparison between studies done with labeled ANIT of different specific activities. Control and cycloheximide- treated groups were compared statistically by the Student’s t test, using a P < 0.05 for rejection of the null hypothesis.

RESULTS

In Experiment 1, we measured the tissue distribution of 14C-ANIT over a 12 hr period. Table I gives the ANIT-equivalents found in blood, liver and fat at blood and the liver followed a rather irregular pattern, with high values found various times after ANIT. In control animals, the concentrations of ANIT in the at 0.5, 1, 4, and 12 hr after ANIT, whereas at 0.25, 2, and 8 hr ANIT con- centrations were lower. A similar pattern was seen in kidney, heart and lungs, where ANIT-equivalents ranged from 40 to 135 pg/g wet wt (data not shown). In fat, a somewhat different pattern was observed: very high amounts of ANIT- derived radioactivity were found 4 and 8 hr after ANIT; later they seemed to decrease. This could indicate that fat might possibly serve as a temporary storage site for ANIT.

The irregular pattern of ANIT-derived radioactivity observed in the control animals was roughly paralleled in the animals pretreated with cycloheximide. However, it seemed clear that in cycloheximide treated animals there was at any given time considerably less radioactive material found in the blood and the various organs examined. The experiment could indicate that treatment with

240 LOCK ET AL.

TABLE I

DISTRIBUTION OF W-ANIT IN CONTROL RATS AND RATS TREATED WITH CYCLOHEXIMIDE'

Time after ANIT-equivalents in ANIT-equivalents in ANIT-equivalents in “C ANIT bloodbJ (pg/ml) liverb.d (pg/g) fatbsd b-g/g)

(h) Controls Cyclo- Controls Cyclo- Controls Cyclo-

heximide heximide heximide

0.25 (6/6)” 31 f 5 13 It 2” 151 i 16 68 f 4e 16f 1 11 f 1” 0.50 (3/3) 88 f 13 12 f 9” 217 f 23 81 f 20” 80 f 15 16 f 3” 1 (3/3) 56 zk 11 21 f 3” 217 f 54 7ozlz 5 170 zk 26 23 f 4” 2 (3/3) 39f 6 30 f 3 154 f 18 112 f 14 184 f 43 32 f 4” 4 (3/3) 92 f 36 34 f 4 248 f 88 96 f 12 473 f 315 72 f 16 8 (3/3) 54f 7 22 f 4” 158 + 18 78 f 126 420 f 55 142 f 24”

12 (3/3) 94 f 12 40 f 6’ 189 f 23 99f 1’ 208 f 26 105 f 24e

a Animals were given cycloheximide (2 mg/kg) or 0.9% NaCl (controls) and, 30 min later, W-ANIT (300 mg/kg, 10 &X/kg).

b dpm converted into pg ANIT-equivalents. c Denotes : 6 controls, 6 cycloheximide-treated animals. d Mean i SE. e Significantly lower (P < 0.05) than control values.

cycloheximide reduces the amount of ANIT passing from the gut into the target organ, the liver.

In Experiment 2, we examined how long a pretreatment time with cyclo- heximide would affect ANIT distribution 1 hr after its administration. This experiment was necessary because it had been found earlier that cycloheximide is capable of protecting against ANIT-induced hyperbilirubinemia even if given 24 hr before ANIT ( Indocochea-Redmond et al., 1973). It became apparent that cycloheximide treatment again resulted in substantially reduced amounts of ANIT-equivalents in all tissues examined, even if cycloheximide was given 24 hr before ANIT (Table II).

The studies of Capizzo and Roberts (1970; 1971a; 1971b) on YZ-ANIT dis- position show that about 7-3s of the total amount of radioactivity administered is eliminated as L4CO, within the first 12 hr. This provides direct evidence for the existence of ANIT biotransformation, although the events leading to the cleavage of the carbon label in the isothiocyanate moiety from the naphthalene ring are not yet elucida,ted. This implies that measuring 14C activity in blood and different tissues can be misleading as long as the products associated with the radioactive label have not been identified as ANIT or a metabolite thereof. Isolation and characterization of such metabolites has repeatedly been attempted, but so far without success (Capizu, and Roberts, 1971a; 1971b; Roberts, 1973). We have also been unsuccessful in this regard. Also, experiments using ANIT labeled with 14C in the isothiocyanate moiety do not give information on the metabolic fate of the naphthalene ring, should it indeed become dissociated. Therefore, we repeated some experiments with ANIT labeled with 3H in position 4 of the naphthalene nucleus ( Fig. 1) .

It was established in several preliminary experiments that 3H-ANIT did not exchange tritium either in vitro or in uioo. In Experiment 3, a distribu- tion study with 3H-ANIT similar to Experiment 1 was performed. The data

CYCLOHEXIMIDE AND ANIT DISTRIBUTION 241

(Table III) g a ain showed two things: in control animals, the concentrations of ANIT in blood and liver followed an irregular pattern, whereas in fat some storage seemed to occur between 4 and 8 hr after ANIT. Animals pretreated with cycloheximide followed the same pattern as controls, except that again at any given time less radioactivity was present. Conversion of the radioactivity into rg ANIT-equivalents revealed that practically identical amounts were found in blood and liver, whether W- or 3H-labeled ANIT was used. However, this was not so in fat: in this tissue, much more 3H-labeled material accumulated between 1 and 4 hr than was found in the experiments using 14C-ANIT. This difference pointed to the possibility that indeed the naphthalene ring might undergo a different disposition than the entire molecule or the isothiocyanate moiety.

It was decided to investigate further whether we could obtain evidence for this not only in fat, but directly in the target organ of ANIT, the liver. A pos- sible way to do this was as follows. Cold ANIT was mixed with W-labeled and 3H-labe1ed ANIT and this mixture was administered to control and cyclohex- imide-treated rats. If ANIT does not undergo biotransformation at all, then one could anticipate that the ratio of 3H/14C activity at any given time point in liver and other tissues would be identical to the “H/W ratio of the administered ANIT. If, on the other hand, loss of the isothiocyanate moiety from the naphtha- lene ring or a carbon-nitrogen cleavage would be a prominent feature in ANIT metabolism, the ratio should change. Moreover, the ratios would possibly show a distinctive pattern in control animals; if cycloheximide was going to affect ANIT metabolism, then the pattern of the 3H/14C ratio might be different from the one found in the controls.

In order to decrease the uncertainty introduced by variations between in- dividual animals, it seemed desirable to follow the sH/14C ratio over a certain

TABLE II

DISTRIBUTION OF I%-ANIT IN CONTROL RATS AND AFTER VARIOUS

PRETREATMENT TIMES WITH CYCLOHEXIMIDF

Pretreatment ANIT-equivalents in ANIT-equivalents in ANIT-equivalents in time before bloodhgd @g/ml) 1iveW &g/g) fatb*d hg/g) 1% ANIT -

(h) Controls Cyclo- Controls Cyclo- Controls Cyclo- heximide heximide heximide

0.50 (3/3)” 56 f 11 21 f 3’ 217 i 54 70f 5 170 f 20 26 f 48 1 F/6) 80 f 3 44 f 5” 224 f 19 146 f 15” 325 f 130 146 + lSE 2 W/6) 60 f 8 34 f 12 192 f 22 109 f 31 344 f 72 127 f 66 4 W6) 65 f 6 25 f 3” 204 f 19 86 f 9” 270 f 27 80 f 14e 8 e/a 90 f 5 58 f 9” 343 f 47 186 f 318 482 f 31 136 f 50e

24 (6/6) 100 f 6 63 f 7e 317 f 24 215 f lge 548 f 63 239 f 55”

Q Animals were injected with cycloheximide (2 mg/kg) and received between 0.5 to 24 hr after cycloheximide W-ANIT (300 mg/kg, 10 &i/kg) p.o. All animals were killed 1 hr after ANIT and tissue concentrations of 1% were measured.

b dpm converted into pg ANIT-equivalents. c Denotes 3 controls, 3 cycloheximide-treated animals. d Mean f SE. 6 Significantly lower (P < 0.05) than control values.

242 LOCK ET AL.

TABLE III

DISTRIBUTION OF 3H-ANIT IN CONTROL RATS AND CYCLOHEXIMIDE-TREATED RATS-

Time after ANIT-equivalents in ANIT-equivalents in ANIT-equivalents in SH-ANIT bloodbn d (pg/ml) liver6ad &g/g) fatbfd &g/g)

(N Controls Cyclo- Controls Cyclo- Controls Cyclo-

heximide heximide heximide -

0.25 (3/3)” 26 f 8 S&2 137 f 26 68 f 11 63f 4 58& 5 0.50 (3/3) 45f 4 13 f 3” 150 f 20 89 f 26 99* 4 69f 5e 1 (3/3) 52 f 15 7 f 1e 197 f 45 56 f 6e 314 f 92 69zk 7 2 (3/3) 43z!z 6 7 zk 3” 161 zk 12 51 f 7” 729 & 209 121 f 12” 4 (3/3) 69f 9 17 f 36 208 f 26 80 f 3” 657 f 91 178 f 37” 8 (4/2) 3?& 2 19 f 3” 122 f 6 74* 5” 364 f 29 233 zk 4”

12 (3/3) 56& 5 16 f le 154 f 14 64 f 6” 282 f 37 127 f 18”

Q Animals were given cycloheximide (2 mg/kg) or 0.9% NaCl (controls) and, 30 min later, aH-ANIT (300 mg/kg, 50 pCi/kg) .

b dpm converted into a ANIT-equivalents. 6 Denotes : 3 controls, 3 experimental animals. d Mean f SE. 8 Significantly lower (P < 0.05) than control values.

time period in the same animal. Multiple organ biopsies were not possible, but it was felt that measurements on blood and bile might give some information concerning the metabolic fate of ANIT. This was done by cannulating the carotid artery and the common bile duct in anesthetized rats; after the adminis- tration of dually labeled ANIT, blood and bile samples were periodically col- lected and analyzed for 3H and 14C radioactivity.

The data are presented in Fig. 2. The ratio of 3H/14C in the ANIT soIution was determined to be 6.3. It is evident from Fig. 2 that, in blood, this exact ratio was never found; the ratio was closest to 6.3 a few minutes after ad- ministration, dropped between 30 and 60 min and seemed to reach a constant value near 5.8 from 1.5 to 8 hr after administration. No significant difference was found between control animals and cycloheximide treated animals. How- ever, highly significant differences between control and cycloheximide treated animaIs were found in bile. In control animals, the 3H/14C ratio stayed close to the value of 6.3 during the first 30 min but then rose up to a value of 7.5 in- dicating that proportionally more 3H was excreted than 14C. In the cyclohexi- mide-treated animals, during the entire experiment the ratio was practically identical to the theoretical ratio of 6.3. Therefore, a qualitative difference in excretion of radioactivity was observed between normal rats and rats treated with cycloheximide. Finally, the cumulative excretion of sH and 14C over the entire 8 hr experimental period was calculated for the experiments represented in Fig. 2. Control animals excreted 1.95% of the administered 3H and 1.770 of the administered 14C in their bile. In cycloheximide treated animals, 0.8Oy’ of SH and 0.79% of 14C were excreted in the bile over the same time period. In both control and treated animals, bile flow was identical #over the entire observa- tion period.

We also analyzed, at various times after the administration of dually labeled ANIT, the 3H/14C ratios in the liver, fat and several other tissues of animals

CYCLOHEXIMIDE AND ANIT DISTRIBUTION 243

I I 2 3

/ 4 5 1

TIME AFTER ANIT ADMINISTRATION (hr.)

FIG. 2. Ratio of ‘H/W in blood and bile after administration of dually labeled ANIT. Rats were injected with cycloheximide (2 mg/kg) or 0.9% NaCl (controls). Thirty minutes later they were given 300 mg/kg of ANIT, labeled either with l’C or “H; the ratio of 8H/z*C was 6.3. At several times after the ANIT, the ratio ‘H/W was determined in bile and blood samples. Each point represents the mean * SE of 4 animals for bile samples, and 2 animals for the blood samples. Values statistically different from controls are marked with an asterisk. n--n, bile: ANIT alone (controls); A-A, bile: cycloheximide and ANIT; O-0, blood: ANIT alone (controls); O-0, blood: cycloheximide and ANIT.

pretreated with cycloheximide and compared them to the corresponding ratios found in control animals (data not shown). It was found that the mean 3H/14C ratio varied with time and that the pattern was not identical in the control ani- mals to that found in the cycloheximide treated rats. However, to obtain tissue

samples for each time point, several animals had to be killed at each time, and rather large variations were observed from animal to animal, especially during

the first hours after ANIT; conclusive interpretation of these data was not possible.

DISCUSSION

It was the aim of the present investigation to obtain some answers why

cycloheximide protects rats against ANIT-induced hyperbilirubinemia and cholestasis. Cap&o and Roberts (1971b) had shown that actinomycin D, which also affords protection against ANIT-induced hepatotoxicity, reduces the accumulation of ANIT in the liver. In the case of cycloheximide pretreatment, the differences in the amount of ANIT-derived radioactivity in blood, liver, and several other tissues found to exist between treated and control animals were substantial throughout. Therefore, it might be concluded that cycloheximide protects simply by reducing the amount of ANIT which eventually reaches the liver.

However, it has been found in other studies that cycloheximide protects only against some, but by no means against all the pathological sequelae of ANIT

244 LOCK ET AL,

hepatotoxicity (Indacochea-Redmond et al., 1974; Traiger et al., 1974). This could indicate that the hyperbilirubinemia and cholestasis produced by ANIT, depend, apparently, not so much upon the amount of ANIT in the liver, but possibly how the liver, or even some other organ, handles ANIT. There are several lines of circumstantial evidence which suggest that metabolic conversion of ANIT into a toxic metabolite seems to be essential for the production of hyperbilirubinemia and cholestasis (Plaa, 1970; Capizzo and Roberts, 1971b). However, in several studies with 14C-ANIT, it has not yet been possible to elucidate and characterize the structure of any ANIT-metabolite (Capizzo and Roberts, 1971a; 1971b; Roberts, 1973; our own observations). The data presented in Fig. 2 of this paper seem to offer a tentative explanation why this has been so and also point out a possible new approach for future studies.

By measuring the 3H/14C ratio in the bile of animals given a mixture of 3H-ANIT and 14C-ANIT, it was found that a highly significant difference exists between control animals and protected animals. The simplest interpreta- tion of the data presented in Fig. 2 is as follows: in normal rats, within I hr or so after ANIT, one or several labeled, ANIT-derived moieties appear in the bile. They appear to contain more 3H relative to 14C than the initially ad- ministered dually labeled ANIT. In other words, the bile might contain a greater amount of ring labeled material than material labeled in both the naphthalene ring and the isothiocyanate moiety. This could mean that normal animals convert some ANIT into a compound consisting of the (modified) naphthalene ring only. This unidentified and still hypothetical “toxic metabolite” would be excreted in the bile, become reabsorbed via the enterohepatic circulation and then might produce liver injury. The observation that bile duct cannulation protects animals fully against ANIT hepatotoxicity (Roberts and Plaa, 1966) is consistent with such a hypothesis. On the other hand, cycloheximide-protected animals excrete material in their bile in which the 3H/14C ratio does not differ from the originally administered material. In other words, protected animals might excrete only unaltered ANIT in their bile. It is important to note that the total cumulative excretion of both 3H and 14C in the bile was identical in the cycloheximide-treated rats, whereas normal animals excreted, on a cumulative basis, more 3H than 14C; these findings again suggest that protected rats excrete unaltered ANIT in their bile and not, as normal animals seem to do, small amounts of a modified ANIT moiety.

It is our current working hypothesis that cycloheximide protects rats by block- ing the conversion of ANIT into a hepatotoxic compound, It would seem that bile is *the most promising b’ody fluid in which to search and from where to isolate the metabolite( s). Hopefully, future studies will not only elucidate its exact chemical structure, but also provide some information why cycloheximide pre- vents its formation.

ACKNOWLEDGMENTS

Expert technical assistance was provided by Miss Suzanne Leroux and Mr. G&&l M&d. This work was supported by the Medical Research Council.

REFERENCES

CAPIZZO, F., and ROBERTS, R. J. ( 1970). Disposition of the hepatotoxin a-naphthylisothio- cyanate ( ANlT) in the rat. Toxicol. Appl. Pharmaco~. 17, 262-271.

CYCLOHEXIMIDE AND ANIT DISTRIBUTION 245

CAPIZZO, F., and ROBERTS, R. J. (1971a). a-naphthylisot,hiocyanate (ANIT) -induced hepato- toxicity and disposition in various species. Toxicol. Appl. Pharmacol. 19, 176-187.

CAPIZZO, F., and ROBERTS, R. J. (1971b). Effect of phenobarbital, chlorpromazine, actino- mycin D and chronic a-naphthylisothiocyanate administration on or-naphthylisothiocyanate- 14C disposition and or-naphthylisothiocyanate-induced hyperbilirubinemia. J. Pharmacol. Exp. Ther. 179, 455-464.

INDACOCHEA-REDMOND, N., WITSCHI, H. P., and PLM, G. L. (1973). Effect of inhibitors of protein and ribonucleic acid synthesis on the hyperbilirubinemia and cholestasis produced by or-naphthylisothiocyanate. 1. Phurmacol. Exp. Ther. 184, 780-786.

INDACOCHEA-REDMOND, N., Wrrscnr, H. P., and PLAA, G. L. (1974). Effects of inhibitors of protein and ribonucleic acid synthesis on a-naphthylisothiocyanate-induced hyper- bilirubinemia, sulfobromophtalein retention and prolongation of pentobarbital hypnosis. J. Phurmacol. Exp. The?. 189,278-284.

PLAA, G. L. ( 1970). Hyperbilirubinemia and cholestasis, a different form of liver injury produced in animals. Essays in Toxicol. 2, 137-154.

ROBERTS, R. J. ( 1973). Microsomal metabolism of the hepatotoxin a-naphthylisothiocyanate (ANIT) following phenobarbital, chlorpromazine or actinomycin D treatment. Proc. Sot. Exp. Bid. Med. 142, 365-367.

ROBERTS, R. J., and PLAA, G. L. ( 1966). The effects of bile duct ligation, bile duct can- nulation and hypothermia on a-naphthylisothiocyanate-induced hyperbilirubinemia and cholestasis in rats. Gastroenterology 50, 768-774.

TRAIGER, G., DE REPENTIGNY, L., and PLAA, G. L. ( 1974). Effects of inhibitors of protein and ribonucleic acid synthesis on the alteration in biliary bilirubin excretion and non- erythropoietically derived bilirubin synthesis after or-naphthylisothiocyanate administration. Biochem. Pharmucol. (in press ) .

VERBIN, R. S., and FARBER, E. (1967). Effect of cycloheximide on the cell cycle of the crypts of the small intestine of the rat. J. Cell Biol. 35, 649-658.

VERBIN, R. S., GOLDBLA~, P. J., and FARBER, E. (1969). The biochemical pathology of inhibition of protein synthesis in uiuo. The effects of cycloheximide on hepatic parenchymal cell ultrastructure. Lab. Inuest. 20, 529-536.

Wrrscr-n, H. P. ( 1973). Qualitative and quantitative aspects of the biosynthesis of ribonucleic acid and protein in the liver and the lung of the Syrian Golden Hamster. Biochem. I. 136. 781-788.