Interactions between bromobenzene dose, glutathione concentrations, and organ toxicities in single- and multiple-treatment studies

FUNDAMENTAL AND APPLIED TOXlCDLDGY 4, 1019-1028 (1984)

Interactions between Bromobenzene Dose, Glutathione Concentrations, and Organ Toxicities in Single- and Multiple-Treatment Studies WILLIAM

M. KLUWE,’

ROBERT R. MARONPOT, ARNOLD GREENWELL, AND FRANK HARRINGTON

National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709 Interactions between Bromobenzene Dose, Glutathione Concentrations, and Organ Toxicities in Single- and Mdtiple-Treatment Studies. KLUWE, W. M., MARONPDT, R. R., GREENWELL, A., AND HARRINGTON, F. (1984). Fundam. Appl. Toxicol. 4, 1019-1028. A single oral dose of 4.0 mmol/kg bromobenzene transiently depleted hepatic and renal reduced nonprotein sulthydiyl group (NPS) concentrations, caused hepatocellular necrosis, and increased serum glutamicpyruvic transaminase activity in male Fischer 344 rats. The depletion of NPS had partially reversed by 24 hr, and NFS concentrations were approximately twice normal values by 48 hr post-treatment. When the effects of single and repeated (once daily for 2, 4, or IO days) treatments with 4.0 mmol/kg were compared, it was apparent that the severity of hepatotoxicity lessened and the percentage depletions of hepatic and renal NFS concentrations decreased with increasing length of bromobenzene treatment. There were essentially no signs of toxicity following the tenth treatment with 4.0 mmol/kg. Single-treatment studies indicated the following dose-response: 2.0 mmol/kg bromobenzene depleted liver NF’S and was hepatotoxic, 0.5 mmol/ kg caused a lesser depletion of liver NPS and was not (overtly) hepatotoxic, and 0.0625 mmol/ kg was the maximum dose that did not deplete liver NFS. The responses to single and multiple (ten) treatments with these representative doses were compared. Liver injury was observed after a single but not after the tenth daily treatment with 2.0 mmol/kg. Both the single and the tenth administrations of 2.0 mmol/kg depleted hepatic NPS, but the prccentage of depletion was greater after the first than after the tenth dose. Liver injury was not detected with lower dose regimens. The patterns of NPS depletion in liver and kidney were, similar alter single or muliple (ten) treatments. The minimum NPS concentrations produced, however, were lower alter single than after multiple treatments. The molar amounts of liver NPS depleted after the tenth treatment appeared to be equivalent to or greater than those after the first, but prior bromobenzene exposure resulted in a higher concentration of tissue NPS being present at the time of the final treatment. Thus, the minimum tissue concentrations of NFS were greater after multiple treatments than after single treatments, despite the loss of equivalent amounts of NPS. It is concluded from these studies that repeated treatment produces resistance to bromobenzene hepatotoxicity. This protective adaptation may be due to a chemically induced increase in liver glutathione concentration. 0 1984 Society of Toxicology.

It is a well-accepted practice in toxicity testing to choose doses for subchronic and chronic animal studies on the basis of doseresponse in preceding acute and subchronic studies (Sontag et al., 1976; Food Safety Council, 1980; Robens et al., 1982). This methodology assumes that animals become no less susceptible and, in fact, may become more susceptible to chemical toxicity upon ’ To whom correspondence should be addressed.

repeated exposure. As our knowledge of mechanisms of chemical toxicity expands, however, the importance of such modulating factors as age, environmental factors, concomitant chemical exposures, and induction or inhibition of xenobiotic metabolism on the toxic response must be considered in toxicity testing. Several chemicals are believed to become proximate toxicants following metabolism to electrophilic intermediates. Many of these 1019

0272-0590/84 $3.00 Copyri%t Q 1984 by the society of Toxicology. All rights of reproduction in any fom reserved.

1020

KLUWE

are detoxified via coniugation with glutathi_one, an abundant cellular nucleophile (Chasseaud, 1976; Mitchell et al., 1976). Not surpringly, therefore, such electrophilic intermediates commonly produce acute tissue injury only when doses sufficient to deplete glutathione concentration in the target tissue below a “critical” level are administered (Mitchell et al., 1973, 1976; Docks and Krishna, 1976; Kluwe, 198 la). Much less is known about the relationshios bcween repeated or chronic chemical administration, dose, glutathione concentration, and toxicity. Several studies have demonstrated a period of increased tissue glutathione concentration following chemically induced depletion (Lambert and Thorgeirsson, 1976; Szabo et al., 1977; Kluwe et al., 1982a,b). One might expect an attenuated toxic response if a subsequent chemical treatment was administered during this time of increased glutathione concentration. The following studies were performed to explore the interrelationships between chemical dose, glutathione depletion, and toxicity in repeated-treatment situations. Bromobenzene was chosen as a model compound for these studies because of the comprehensive data base relating its acute toxicity to glutathione depletion (Jollow et al., 1974; Thor et al., 1978, 1982; Pessayre, 1979. METHODS General. Adult (12-16 weeks of age) male Fischer 344’ rats were housed in “shoe-box”-type polycarbonate cagesand maintained in temperature (22-24”(Z), humidity (40-60%, relative), and light-cycle (12-hr thtorescent light, 12 hr dark) controlled rooms throughout the experiment. Food3 and water were provided ad libitum. Bromobenzen$ (greater than 98% purity) was mixed with corn oils and administered by gavage with 4-in., round-tipped feeding needles6 The total volume admin* Charles River Breeding Laboratories, Portage, Mich. ’ NIH-31 diet, Ziegler Brothers, Gardner, Pa. ’ Aldrich Chemical Company, Milwaukee, Wise. ’ Laboratory Grade, Fisher Scientific, Fairlawn, N.J. 6 H. Popper and Sons, Inc., New York.

ET AL. istered was 0.20 ml/100 g body wt. All bromobenzene treatments were given between 0900 and 1000 hr to control for diurnal variation in tissue nonprotein sulthy-

dVl (Nps) concentrations.

Comuarative rewonse to sinnle and multiule treatment with 4.0 mmol/kg bromobenzene. One hundred seventysix rats were divided into groups of four animals and treated once daily for I, 2, 4, or 10 consecutive days with 0.0 mmol/kg (vehicle control) or 4.0 mmol/kg (628 mg/kg) of bromobenzene. Equal numbers (N = 4) of animals from each treatment group were killed by CO* asphyxiation exactly 3, 6, 9, 12, or 24 hr after the final treatment. Animals from the I- and IO-day groups were also killed 48 hr after the final treatment. Livers and kidneys were quickly removed and weighed. Samples were homogenized in 15 (liver) or 9 (kidneys) vol of 3% trichloroacetic acid-l mM EDTA at 4°C. The precipitate was removed by centrifugation and NPS concentrations were measured in the supematant fraction using Ellman’s reagent (Ellman, 1959). Reduced glutathione’ was used to prepare the standard curve. For animals killed 24 hr after the final treatment, venous blood was collected under light CO2 anesthesia from an orbital sinus immediately prior to killing. Serum was separated and analyzed for ghttamic-pyruvic tram+ aminase (GPT) activity, sorbitol dehydrogenase (SDH) activity, urea nitrogen concentration (BUN), and creatinine concentration using spectrophotometric techniques (Kluwe, I98 1b). Samples of liver and kidney were fixed in neutral buffered 10% Formalin, embedded in paraKin, sectioned at 6 pm, and stained with hematoxylin and eosin. All of the tissue sections were analyzed microscopically in a blind fashion. Diagnoses were assigned, and lesion severities were graded according to the specifications listed beneath the appropriate tables. Dose-response for single bromobenzene treatments. One hundred twenty-eight rats were divided into groups of four animals and treated once, by gavage, with 0.0 (vehicle control), 0.0625, 0.25, 0.50, 1.0, 2.0, 3.0, or 4.0 mmol/kg bromobenzene. Four rats from each dose group were killed 3, 6, 9, or 24 hr later. NPS concentrations (ah sacrifice times), serum chemistries (24 hr only), and liver and kidney histopathologies (24 hr only) were evaluated as described in the preceding paragraphs. Comparative dose-response for single and multiple bromobenzene treatments. Ninety-six rats were divided into groups of six animals and received ten daily treatments with 0.0 (vehicle control), 0.0625, 0.50, or 2.0 mmol/kg bromobenzene. Another 48 rats were divided into groups of three animals and received a single treatment with the same doses. The treatments were timed so that all of the animals received the final treatment on the same day. The animals were killed 3, 6,9, or 24 hr aher the final treatment. NPS concentrations (all sacrifice times), serum chemistries (24 hr only), and ’ Sigma Chemical Company, St. Louis, MO.

BROMOBENZENE liver and kidney histopathologies (24 hr only) were evaluated as described in the preceding paragraphs. The molar amounts of hepatic NPS depleted or otherwise lost aher the first and tenth treatments with bromobenzene were estimated using group mean pretreatment (time zero) tissue NPS concentrations and group mean liver weights. For those animals receiving ten daily bromobenzene treatments, liver NPS concentrations 24 hr after the ninth treatment (time zero for the tenth treatment) were assumed to be the same as those 24 hr after the tenth treatment. Statistical analyses. The data were analyzed by analysis of variance, completely randomized design, and treatment means were compared to the appropriate control with Student’s t test (Sokal and Roblf, 1969). In all instances, treated groups were compared only to vehicle controls receiving the same number of treatments. For comparisons of NPS concentrations, the data were initially analyzed using the measured tissue concentrations (micromoles of NPS per gram of tissue); the data were then converted to percentages of control for graphical purposes (influence of diurnal variation removed). All other data were analyzed and presented as the actual quantities measured.

RESULTS The diurnal variation in hepatic NPS concentration is illustrated in Fig. 1. Neither single nor repeated treatments with the corn oil vehicle (0.2 ml/100 g body wt) depleted hepatic NPS or affected the diurnal pattern

Time of day

I. Diurnal variation in liver and kidney nonprotein sulfhydryl concentration. Rats were killed at the times indicated and nonprotein sulthydryl (NPS) concentrations were measured in the liver and kidneys. Each symbol represents the 2 + 1 SE of four animals. FIG.

AND GLUTATHIONE

1021

of tissue NPS concentrations. No remarkable diurnal variations in renal NPS concentrations were observed. Comparative Response to Single and Multiple Treatments with 4.0 mmol/kg Bromobenzene A single treatment with 4.0 mmol/kg bromobenzene depleted both hepatic and renal NPS, the lowest concentrations being reached 3-12 hr post-treatment (Fig. 2). The magnitude of the depleting effect was much greater in the liver than in the kidneys. Tissue NPS concentrations increased thereafter and were markedly elevated above normal 48 hr posttreatment. Depletions of renal NPS below normal concentrations did not occur following multiple treatments with 4.0 mmol/kg bromobenzene. (Although a further decline between 12 and 24 hr cannot be ruled out, the lack of a change in NPS concentration between 9 and 12 hr suggests that the minimum concentration had been achieved.) The minimum glutathione concentration in the liver increased in a manner roughly proportional with the length of treatment and was virtually the same as in the controls (near 100%) after the tenth treatment (Fig. 2). Neither liver nor kidney weight (expressed relative to body weight) was increased significantly by single or multiple bromobenzene treatments (Table 1). Serum GPT activity was increased at the first, second, and fourth administrations of 4.0 mmol/kg bromobenzene, but not after the tenth administration. Moreover, the increase in serum GPT activity was highest 24 hr after the second treatment. No significant increases in BUN or serum creatinine concentrations were observed, although one of the four animals receiving 4.0 mmol/kg for 10 days exhibited abnormally high values (BUN, 146 mg/dl, creatinine, 3.44 mg/dl). Within 24 hr, a single dose of 4.0 mmol/ kg bromobenzene produced focal centrilobular and midzonal hepatocellular necrosis of

1022

= 4

KLUWE

40

tI

I 3

I 6

I I If 9 I2 ” Hours post-treatment

8 24

I 48

FIG. 2. Depletion of hepatic and renal nonprotein sulthydryl contents following single or repeated treatments with 4.0 mmol/kg bromobenzene. Rats were killed at the indicated times following one, two, four, or ten daily treatments with 0 (vehicle control) or 4.0 mmol/kg bromobenzene. Nonprotein sulthydryl concentrations were measured in the liver and kidneys and presented as percentages of control. Each symbol represents the X f 1 SE of four animals; open symbols are significantly different from controls, p < 0.05. Control values for hepatic NPS concentrations (cmol/g tissue) were as follows for 3, 6, 9, 12, 24, and 48 hr after treatment, respectively: single exposure-4.8 1 + 0.46, 4.03 + 0.27, 3.28 f 0.22, 3.46 f 0.12, 6.06 + 0.14, 5.16 of:0.31; two exposures-5.45

f 0.14, 6.30 kO.16,

4.33

+ 0.15, 4.97 + 0.50, 4.59 +- 0.17, 3.46 + 0.17 (no 48 hr); four exposures-l.92 k 0.05, 3.98 + 0.12, 3.28 -t 0.12, 5.31

f 0.17 (no 48 hr); ten exposureH.72

-t 0.35, 4.34

f 0.34,

-t 0.28,

3.53

+ 0.14,

3.13

+ 0.14,

5.77

5.10

+- 0.33. Renal NFS concentrations did not change with time of day and remained within a range of 2.36-2.88 pmol/g tissue in all control groups.

moderate severity (+3) on a scale: 0 (none), +l (minimal), +2 (mild), +3 (moderate), +4 (severe), with an attendant mononuclear inflammatory response. Periportal hydropic change characterized by clear cytoplasmic areas was also observed (Table 2). The hepatocellular necrosis and inflammatory re-

ET AL.

sponse were somewhat more severe (+4) 24 hr after the second treatment, and the mitotic index in the midzonal and periportal areas was increased at this time (indicating cell division). Hepatocellular necrosis was minimal 24 hr after the fourth treatment and absent 24 hr after the tenth treatment with 4.0 mmol/ kg bromobenzene (Table 2). Granulomatous hepatitis (mononuclear cells, histiocytes, giant cells) and focal calcification in the centrilobular area, presumed secondary to hepatocellular necrosis, were observed after the fourth treatment, but not the tenth. Periportal and midzonal cytoplasmic vacuolation, probably glycogen deposits, were observed after four and ten treatments. No liver lesions were observed in any of the corn oil (vehicle)treated rats. The kidneys from all rats were normal in appearance, except for two animals that received ten treatments with 4.0 mmol/kg. One of these exhibited moderate tubular dilatation, and the other (the one with elevated BUN and creatinine values) severe necrosis of -30% of the convoluted tubules. Dose-Response Treatments

for

Single

Bromobenzene

Dose-dependent depletion of renal and hepatic NPS by single treatments with bromobenzene is illustrated in Fig. 3. Doses of 4.0, 3.0, or 2.0 mmol/kg caused similar initial degrees of hepatic Nps loss, although recovery was somewhat more rapid after 2.0 mmol/ kg. Both 0.50 and 0.25 mmol/kg also caused hepatic NPS depletion, while 0.0625 mmol/ kg was without effect. (A dose of 0.125 mmol/kg caused equivocal changes in hepatic NPS concentration.) Bromobenzene had lesser effects on renal than on hepatic NPS; depletions of renal NPS were not observed at 0.50 mmol/kg or lower doses (Fig. 3). Some rats receiving 1.0 mmol/kg bromobenzene had slightly elevated serum activities of GPT or SDH 24 hr post-treatment, but

BROMOBENZENE

AND TABLE

1023

GLUTATHIONE

1

RESPONSETO SINGLE OR MULTIPLE TREATMENTS WITH 4.0 mmolFg BROMOBENZENE No. of bromobenzene treatments Parameter Liver/body weight (XlW GPT (W/L) SDH (W/L)

Kidneys/body weight WlW Creatinine 0w.U BUN (mf$dl)

Control a

1

2

4

10

4.96 zk 0.36 52 k 6 30 + 6

4.54 + 0.36 410 f 250b ND

5.06 + 0.49 3618 + 2070’ ND

5.21 + 0.26 221 f 69’ 60 + 6b

5.28 f 0.71 62 T!Z14 25 f 14

0.90 + 0.05

0.89 + 0.06

0.87 k 0.04

0.89 + 0.02

0.91 + 0.08

0.68 f 0.03

0.65 t- 0.05 24 + 1

0.54 rt 0.05 29 + 7

0.69 k 0.12

1.36 + 1.38’ 53 + 62’

21*2

19 + 1

Note. Rats were killed 24 hr after the final treatment. Each value is the X + SE of four animals. GPT, glutamicpyruvic transaminase activity; SDH, sorbitol dehydrogenase activity; BUN, urea nitrogen concentration; ND, not determined. ‘Representative values. For statistical analyses, each bromobenzene-treated group was compared to a control group receiving the same number of vehicle treatments. b Si@icantly greater than appropriate control group, p i 0.05. c Large variability due to a single animal with hi8h values.

TABLE

2

SUMMARYOFHISTOPATHOLOGICCHANGESINTHE LIVER IN RESPONSETO SINGLE ORMULTIPLE TREATMENTS WITH 4.0 mmol/kp BROMOBENZENE Incidence (severity)”

Treatment group

Hepatocellular necrosis

Inflammatory response

Hydropic change (peripoltal or OlidZOMl)

Increasedmimic index (periportal or midmnaI)b

cytop*c vacuolization (peripo~ 01 midzonal)

Control, single treatment Bmmobenzene,SingIe treatment

O/4

O/4

O/4

O/4

O/4

4/4 (+3)

414 (+3)

414 (+2)

O/4

O/4

two treatments Bmmobenzene,two treatments

O/4

Of4

O/4

Of4

O/4

414 (+4)

414 (+4)

214 (+2)

414

O/4

Control, four treatments Bromobenzene,four treatments

O/4

O/4

O/4

O/4

O/4

414 (+I)

414 (+3)

O/4

114

414 (+3)

Control, ten treatments Bmmobenzene,ten treatments

O/4

O/4

O/4

O/4

O/4

O/4

O/4

O/4

O/4

314 (+4)

Control,

Nose.The rats were killed 24 hr afkr the final treatment. * Severity of the lesions was graded as follows: 0, absent; + 1, minimal, b Severity of lesions was not graded.

+2, mild; +3, moderate;

+4, marked or severe.

1024

=k izof IOO‘s 8 : BOt f 60I” 4020-

1000 =8 Bo: 60H 5 40-s $2 20-

KLUWE

II :n

FIG. 3. Dose- and time-dependent depletion of hepatic and renal nonprotein sultbydryl contents by single administrations of bromobenzene. The rats were killed at various times following a single administration of 0 (vehicle control), 0.0625, 0.25, 0.5, 1.0, 2.0, 3.0, or 4.0 mmol/kg bromobenzene. Nonprotein sulthydryl contents were measured in the liver and kidneys and presented as percentages of control. Each bar represents the k + 1 SE of four animals; open bars are significantly different from control, p < 0.05. Control values for hepatic NPS concentrations (pmol/g tissue) were as follows for 3, 6, 9 and 24 hr alter treatment respectively: 4.52 f 0.19, 3.55 + 0.53, 2.95 + 0.15, 5.02 + 0.19. Renal NPS concentrations did not change with time of day and remained within a range of 2.66-2.86 amol/g tissue.

the group mean values were significantly increased only at the 2.0, 3.0 and 4.0 mmol/ kg doses (Table 3). Relative liver weights were reduced at 3.0 and 4.0 mmol/kg. No clinically significant increases in serum creatinine or BUN concentrations were observed. Although of statistical significance, the minor elevations of creatinine concentration occurring at 2.0-4.0 mmol/kg were not considered to be clinical evidence of nephrotoxicity, based on the authors’ historical

ET AL.

experience with this rat strain and the lack of observed histological abnormalities. Centrilobular and midzonal focal hepatocellular necrosis or degeneration was observed within 24 hr in all rats receiving 2.0, 3.0, or 4.0 mmol/kg bromobenzene, and the severity of the lesion increased with increasing dose. The predominant morphologic lesion at 1.0 mmol/kg was a periportal and midzonal hydropic change (mild-moderate), while minimal periportal and midzonal cytoplasmic vacuolization was observed in most animals at 0.5 mmol/kg. No renal lesions were observed. Based on these findings, three doses were chosen for the next phase of study: 2.0 mmol/kg, the minimum dose producing both hepatotoxicity and a depletion of hepatic NPS; 0.50 mmol/kg, a dose producing hepatic NPS depletion but not hepatotoxicity; and 0.0625 mmol/kg, a dose without demonstrable effects on the liver. Comparative Dose-Response for Single and Multiple Bromobenzene Treatments Both the 0.50 and 2.0 mmol/kg doses depleted hepatic NPS when administered once or for 10 consecutive days (Fig. 4). The time dependencies of the patterns of NPS concentrations were similar for both singleand multiple-treatment regimens, but the minimum tissue NPS concentration produced was lower after one than after ten treatments. A depletion of renal NPS was observed 6 hr after a single treatment with 2.0 mmol/ kg, but not after ten daily treatments (Fig. 4). Losses of renal NPS were not observed with other treatment regimens. With the exception of some minimal-tomoderate increases in relative liver weights, the only signs of hepatic injury occurred in rats receiving a single treatment with 2.0 mmol/kg bromobenzene; serum GPT and SDH activities were significantly increased (Table 4), and a mild, focal, centrilobular and midzonal hepatocellular necrosis and an

BROMOBENZENE

1025

AND GLUTATHIONE TABLE 3

DOSE-RESPONSETO SINGLE TREATMENTS WITH BROMOBENZENE

Parameter Liver/body WlW

0 (control)

2.0

1.0

0.5

3.0

4.0

weight

GPT (W/L) SDH (W/L) Kidney/body weight (Xl@3 Creatinine (mgjdl)

4.26 f 0.21 59f9 42 f 2

4.54 49 36

+ + +

0.20 3 4

4.91

+ + +

0.89 0.54 16

f 0.04 f 0.02 f 1

0.88 0.58 18f

0.81 0.54 20

BUN bw’~)

0.03 0.02 2

f 0.20" 190 -c 119 105 k 66 + 0.03 f 0.01 I

4.51 220 113

f + +

0.14 24" 14"

3.53 154 68

f 0.15b + 10’ f 13"

0.87 0.55 24

f + +

0.02 0.03 2

0.78 f 0.07 0.63 f 0.04“ 24 f 1

Note. The rats were killed 24 hr after treatment. Each value is the X f SE of four animals. GPT, gh~tami~pyruvic activity; SDH, sorbitol dehydrogenase activity; BUN, urea nitrogen concentration. ’ Significantly greater than control, p i 0.05. * Significantly lesser than control, p < 0.05.

inflammatory response were observed. No frank hepatotoxic effects were observed in rats receiving ten daily treatments with 2.0

3.56 184+ 107

f

0.76 0.65 23

+ 0.02 f 0.03' f 1

+

transaminase

mmol/kg, although minimal hydropic change was observed in the periportal and midzonal aspects of the liver lobule. There were no

50 0.0625mrrmVl

t

= f loo 8 3 i2 4

o--

%-

‘---d

so 0.5OmmoMg

P B -i:-

1.50

II

I 3

A

6 9 Hours post-tmtment

f-

0.07* 37" 16"

B

2Ommollkg

36gHours

’

24

post-treatment

FIG. 4. Comparative dose- and time-dependent depletions of hepatic and renal nonprotein sulthydryl contents following one or ten daily treatments with bromobenzene. Rats treated once (closed symbols) or for 10 days (open symbols) were killed 3, 6, 9, or 24 hr after (final) treatment. Nonprotein sulthydryl contents were measured in the liver (A) and kidneys (B) and are presented as percentages of control. Asterisks indicate a significant difference from control, p c 0.05. Each symbol represents the X + 1 SE of six animals. Control values for hepatic NFS concentrations (pmol/g tissue) were as follows for 3, 6, 9, and 24 hr after treatment, respectively: single exposure-4.47 + 0.29, 4.17 + 0.16, 3.17 + 0.19, 3.62 + 0.27; ten exposures-5.09 * 0.69, 4.19 + 0.70, 3.00 f 0.14, 3.85 * 0.26. Renal NFS concentrations did not change with time of day and remained within a range of 2.32-2.87 amol/g tissue.

1026

KLUWE TABLE

ET

AL. 4

DOSE-RESPONSETOSINGLEORMULTIPLETREATMENTSWITHBROMOBENZENE Dose (mmollkg) No.of Treatments

Parameter Liver/body (X100)

weight

GPT W/I)

SDH (W/l)

0 (control)

0.0625

0.50

1 10

3.60 + 0.14 3.74 f 0.16

3.14 + 0.35 3.88 f 0.40

4.19 + 0.26” 4.66 31 0.26’

3.14 f 0.58 4.78 + 0.15@

I 10

37 + 4 35 + 2

36 + 8 34 ?I 5

46 + 5 4Of4

113 f 36” 40 f 46

1

25 t II 22 f 3

17*4 14 f 7

17*4 17 k 6

96 * 17’ 10 f 36

0.74 * 0.04 0.81 + 0.03

0.69 + 0.02 0.86 f 0.06

0.82 + 0.08” 0.74 2 0.02

0.70 * 0.05 0.80 f 0.026

0.71 0.70

0.71 * 0.03 0.74 Ik 0.05

0.74 + 0.02 0.70 + 0.03

0.78 0.68

IO

Kidney/body

weight

WlW

Creatinine

I 10

(mg/dI)

1 10

BUN (mg/dl)

I 10

* 0.01 + 0.06

15 f I 13 + I

13 f 12f

1 I

2.0

I5 * 3 17 i 2

+ 0.03 f o&i*

15 +- 2 12+ lb

No@. The rats were killed 24 hr a&r the final treatment. Each value is the X C SE of three (single treatment) or six (ten treatments) animals. GPT, giutamic+pyruvic transaminase activity; SDH, sorbitol dehydrogenase activity; BUN, urea nitrogen concentration. “Significantly greater than the appropriate control group, p i 0.05. bN=5.

indications of bromobenzene-induced nephrotoxicity in this portion of the study. The amounts (molar equivalents) of hepatic NPS lost by chemical depletion or diurnal variation were estimated using liver weights and pretreatment liver NP!3 concentrations. For animals receiving ten daily treatments, it was assumed that hepatic NPS concentrations at the time of receiving the tenth treatment (24 hr after the ninth treatment) were similar to those 24 hr after the tenth treatment. At any given time after the 0.5 or 2.0 mmol/kg doses, the amounts of hepatic NPS lost (micromoles) after the tenth treatment were equivalent to or greater than that after the first treatment (Table 5). DISCUSSION The data in this study indicate that rats can develop resistance to the hepatotoxic effects of bromobenzene. That is, a dose that was hepatotoxic when administered once did not elicit a hepatotoxic response following

repeated administrations. Such a phenomenon might have been predicted on the basis of an eventual increase in liver NFS concentration following initial depletion. A larger pool of NPS would have to be depleted before tissue injury occurred, and the threshTABLE

5

Dose (mm@W Timeposttreatment (hr)

No. of treattients

0.50

2.0

gmol

NPS lost

3

1 10

68 147

68 71

6

1 10

70 66

91 163

9

1 10

2 3

34 204

BROMOBENZENE

old hepatotoxic dose of bromobenzene would be quantitatively increased. In support of this possible explanation for the developed resistance to bromobenzene, estimates of the amounts of liver NPS lost (Table 5) suggested that the tenth and final bromobenzene treatments depleted as many molar equivalents of NPS as did the first. Therefore, the reduced susceptibility to hepatic injury would not appear to be caused by decreased metabolism of bromobenzene to an electrophilic reactant. At least two other classical hepatotoxicants, carbon tetrachloride (Ccl.+) and ally1 alcohol, have been shown to induce resistance to their own hepatotoxic effects (Glende, 1972; Lake et ul., 1978). The mechanism for resistance to the periportal hepatocellular necrosis normally caused by ally1 alcohol is not known. CCL, which causes a centrilobular hepatocellular necrosis similar to that produced by bromobenzene (Plaa, 1980), is thought to induce autoresistance by destroying the microsomal enzymes necessary for its activation to a toxic molecule (Ugazio, 1972; Lindstrom and Anders, 1977; Guzelian and Swisher, 1979). It is unlikely that tissue glutathione concentration is directly related to Ccl, resistance, since CCL+ does not deplete liver glutathione (Harris and Anders, 1980; W. M. Kluwe, unpublished information). Similarly, it is unlikely that bromobenzene resistance is due to the destruction of microsomal enzymes (although it is activated to a toxic molecule by microsomal systems), since the “nontoxic” tenth treatment would appear to have produced the same amount of NPSdepleting electrophilic intermediate as the first. An alternate explanation, however, is that repeated bromobenzene administration may have enhanced the metabolism of the parent compound to a less-toxic reactive metabolite (e.g., the 2,3-epoxide; Zampaglione et al., 1973; Lau and Zanoni, 1979, 198 1) still capable of conjugating with and depleting glutathione. The role of NPS in modulating the nephrotoxic effects of bromobenzene is less clear

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AND GLUTATHIONE

than those with the liver. Reid (1973) reported kidney injury and renal NPS depletion following single treatments with bromobenzene or chlorobenzene. In the present study, however, a severely hepatotoxic dose (4.0 mmol/ kg) produced littIe evidence of kidney damage and only slight (less than 20%) losses of renal NPS. The only frank evidence of bromobenzene nephrotoxicity in the present study occurred after ten daily treatments with 4.0 mmol/kg. Developed resistance to bromobenzene liver injury, therefore, may not extend to kidney injury. The duration of this study was too brief to explore the limits of developed resistance to bromobenzene hepatotoxicity in terms of either dose or chronicity of treatment. Factors such as animal age, nutritional status, and concurrent disease, or chemical stresses are likely to affect adaptation. These data, however, warn against using acute or subacute toxicity data as the sole determinants of “tolerable” doses for subchronic or chronic studies. Rather, a complete understanding of the interrelationships between chemical dose, chronicity of exposure, and toxic response patterns is needed to fully comprehend toxicity testing results. REFERENCES CHASSEAUD,L. F. (1976). Conjugation with glutathione and mercapturic acid excretion. In Gluruthione: Metabolism and Function (I. W. Aries and W. B. Jakoby, eds.), pp. 357-366. Raven Press, New York. DDCKS, E. L., AND KRISHNA, G. (1976). The role of glutathione in chloroform-induced hepatotoxicity. Exp. Mol. Pathol. 24, 13-22. ELLMAN, G. (1959). Tissue sullhydryl groups. Arch. Biochem. Biophys. 82,70-77. Food Safety Council (1980). Proposed System fir Food Safety Assessment. Food Safety Council, Washington, D.C. GLENDE, E. A., JR. (1972). Carbon tetrachloride-induced protection against carbon tetrachloride toxicity: Role of the liver drug metabolizing system. B&hem. Pharmucoi. 21, 1697-1701. GUZELIAN, P. S., AND SWISHER, R. W. (1979). Degradation of cytochrome P-450 haem by carbon tetrachloride and 2-allyl-2-isopropylacetamide in rat liver in vivo and in vitro. B&hem. J. 184, 481-489.

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