Protein A protects mice from depletion of biotransformation enzymes and mortality induced by Salmonella typhimurium endotoxin

1

Toxicology Letters. 49 (1989) I-13 Elsevier

TOXLET

022 11

Protein A protects mice from depletion of biotransformation enzymes and mortality induced by Salmonella typhimurium endotoxin

P.D. Dwivedi, A.S. Verma, Anil Mishra, K.P. Singh, A.K. Prasad, A.K. Saxena, K.K. Dutta, N. Mathur and P.K. Ray Immunobiology

Division. Industrial Toxicology Research Centre. P.O. Box No. 80, Mahatma Gandhi

Marg, Lucknow 226001 (India) (Received

10 October

(Revision

received 2 May 1989)

(Accepted

3 1 May 1989)

Ke.v words: Protein

1988)

A; Biotransformation

enzymes;

Salmonella typhimurium endotoxin;

Protection

SUMMARY Changes

in hepatic microsomal

demethylase,

glutathione

levels of aspartate

transaminase,

Swiss albino mice exposed Animals

exposed

(5 pg/per

mouse) protected

mixed-function

Stransferase),

glutathione

alanine

oxidase enzyme levels (aniline hydroxylase, content,

transaminase

total sulphydryl

and alkaline

to Salmonella typhimurium endotoxin

to the same dose of endotoxin the animals

but pretreated

from both mortality

content,

phosphatase

(S&l50

were studied

fig per mouse,

with protein

and depletion

aminopyrine

and plasma enzyme in male

LCsO 141.82 fig).

A of Sfuphylococcus

of biotransformation

aureus

enzymes.

INTRODUCTION

Endotoxins, such as that derived from SaImonella typhimurium, can cause a variety of changes in the physiology of an animal. The administration of endotoxin to the mouse increases its body temperature [l] and the levels of free fatty acids and triglycerides. It decreases lipoprotein lipase [2], hepatic cytochrome P-450, aminopyrine demethylase and lipid peroxidase levels [3-51. Administration of bacterial endotoxin (extracted from the cell wall of gram-negative bacteria) lowers mouse serum glucose level and increases levels of serum pyruvate and acid phosphatase, whereas red blood Address

for correspondence:

No. 80, Mahatma

0378-4274/89/$3,50

Gandhi

Marg,

Dr. P.K. Ray, Director, Lucknow

Industrial

Toxicology

Research

226001, India.

@ 1989 Elsevier Science Publishers

B.V. (Biomedical

Division)

Centre,

Post Box

2

cells, haemoglobin and haematocrit values do not seem to change after endotoxin shock [6]. Endotoxin has been shown to be a potent activator for interleukin-1 production from human monocytes [7]. Protein A, a cell wall glycoprotein of Staphylococcus aweus Cowan I, has a multipotent immunopotentiating property [8-lo]. Prior studies showed that protein A, when infused intravenously, can cause regression of primary rat mammary tumours [I I-1 31, dog tumours [14] and mouse tumours [IS]. In the present study, it has been investigated whether protein A can minimise or diminish the toxic effects of endotoxins derived from S. typhimurium, with particular reference to its role in hepatic xenobiotic-metabolising enzymes. Endotoxins are known to decrease the levels of xenobiotic-metabolising enzymes, thereby proving that they are toxic to the liver 13, 4]. As protein A showed abrogation of endotoxin-induced mortality caused by endotoxin in our preliminary studies, it was of interest to know whether protein A was responsible for the change in xenobiotic-metabolizing enzyme levels, thereby reducing the toxicity of endotoxin. MATERIALS

AND METHODS

Animals Male outbred Swiss albino mice (20 &-2 g) obtained from the ITRC animal breeding colony and raised on a commercial pellet diet (Hindustan Lever, Bombay) and water ad libitum were used in the present study. Chemicals Endotoxin extracted from S. typhimurium (LPS) was supplied by Difco Laboratory, U.S.A. Protein A was purchased from Pharmacia Fine Chemicals, Sweden.

The animals were divided into 8 groups of 8 mice each and were treated as follows: Normal saline 0.5 ml i.p. twice weekly for 2 weeks. Group I (Control) Group II Protein A (5 pg per mouse) i.p. twice weekly for 2 weeks. Group III Protein A (5 fig per mouse) i.p. twice weekly for 2 weeks. Endotoxin 50 ,ug in 0.5 ml normal saline i-p. 48 h after the last injection of protein A. Group IV Protein A (5 pg per mouse) i.p. twice weekly for 2 weeks. Endotoxin 100 pg in 0.5 ml normal saline i.p. 48 h after the last injection of protein A. Group V Protein A (5 ,ug per mouse) i.p. twice weekly for 2 weeks. Endotoxin 150 ,ug in 0.5 ml normal saline i.p. 48 h after the last injection of protein A. Group VI Endotoxin 50 ,ug in 0.5 ml normal saline per mouse once i.p.

3

Group VII ~ndotoxin 100 ,ug in 0.5 ml normal saline per mouse once i.p. Group VIII Endotoxin 150 I;tgin 0.5 ml normal saline per mouse once i.p. Preparation

of enzyme source material from mouse liver

The animals were killed 48 hours after the last treatment by cervical dislocation. Livers were removed, blotted free of blood, and washed in ice-cold saline. Tissue homogenates were made in 10% of 0.25 M sucrose. Homogenates were centrifuged at 9000 x g for 20 minutes at 4°C. The resulting supernatant (postmitochondrial supernatant) was used for the measurement of cytochrome P-450 dependent enzymes and glutathione S-transferase (GST) activity. Total sulphydryl (-SH) and glutathione (GSH) contents were measured in tissue homogenate. Co~Iectio~ of blood and separation of plasma

Blood was collected in heparinised test tubes from the retro-orbital plexus of the animals; plasma was separated by centrifugation of blood and was used for estimation of alkaline phosphatase (ALK-P), alanine transaminase (ALT) and aspartate transaminase (AST). Enzyme assays

Aminopyrine demethylase (APD) activity was assayed according to Cochin and Axelrod [ 161. The specific activity was expressed as nanomoles. of formaldehyde fo~ed/min/mg protein. Aniline hydroxylase (AH) activity was assayed by measuring the formation of p-aminophenol according to Kato and Gillette [17] and the activity was expressed as nanomoles of p-aminophenol fo~ed/min/mg protein. GST activity was determined according to the method of Habig et al. [18] using l-chloro2,4_dinitrobenzene (CDNB) as substrate. Total sulphydryl and GSH contents were assayed according to Jollow et al. [19]. AST and ALT in plasma were measured according to the method of Reitman and Frankel [20]. ALK-P in plasma was measured by the method of Wotten [21] using dinitrophenyl phosphate as substrate. Protein content was determined according to Lowry et al. [22], using bovine serum albumin as a reference standard. statistical

analysis

The LCsOvalue of endotoxin was determined by Probit analysis [23]. The effect of different doses of protein A on the toxicity of endotoxin was analysed by using two-way classification with an unequal number of animals in different groups. The model thus used was: Xijk=p++i+Bj+lij+eijk,

where x@ denotes mortality at the ith level of endotoxin and jth level of protein A, and Li and Bi denote the endotoxin and protein A effects, respectively. & represents the interaction between the two factors, ,U the overall mean effect; eijk denotes the independent random error, which is normally distributed at mean zero variance.

4

TABLE

I

DETERMINATION

OF LETHAL

Dose of endotoxin

AND SUBLETHAL

&g)

DOSES OF ENDOTOXIN

S/T (24 h)

S/T (48 h)

0

lo/lo

lo/lo

50

lo/lo

IO/l0

100

s/10

S/IO

200

s/10

2/10

400

2/10

O/IO

S/T = Surviving

animals/total

95% upper confidence

animals;

LCso = 141.82 pg per mouse;

95% lower confidence

= 110.71;

= 18 1.62.

The values presented for enzyme estimation are means f standard errors. Student’s r-test was used for determination of statistical significance. Differences that resulted in probability values smaller than 0.05 were considered to be significant. RESULTS

Determination of lethal and sublethal doses of S. typhimurium endotoxin Fifty mice were divided into 5 equal groups and were given 0, 50, 100, 200 and 400 ,ug of endotoxin per mouse respectively by the intraperitoneal route in 0.5 ml sterile saline (Table I). The control group received only saline. The LC5,, value of en-

q

Untreated

Control

Gr.

61 Gr with

pretreatment

of lug

q

Gr. with

pretreatment

of 2 up

PA.

0

Gr. with

pretreatment

of 5 ,ug PA.

PA

100

200

150 fig

Fig.

1. Effect of varying doses (lOWlO pretreated

Endotoxin

/mouse

pg per mouse) of S. typhimurium

with protein

endotoxin

A (PA) (I,2 and 5 fig doses, respectively).

on mortality

of mice

5

dotoxin

(48 h) was determined

to be 141.82 pg per mouse

limit (95%) of 110.71 and an upper confidence

with a lower confidence

limit of 181.62.

Determination of the EDJO dose of protein A The extent of protection provided by protein A (PA) in mice against endotoxin lethality was estimated by using the latter in doses of 100, 150, 200 and 400 ,ug per mouse respectively in PA-pretreated mice. Mice were divided into 4 groups, viz. control (without pretreatment) and those exposed to 1.O, 2.0 and 5.0 pg PA, respectively (Fig. 1). Survival was recorded for 7 days, but there was no death in any group after 2 days of endotoxin exposure. When 100 pg of endotoxin (En) was administered, the mortality in the untreated group (control) was 25%, whereas in 1 ,ug PA-pretreated (PA + En) animals the mortality was 16.7 % and with 2 pg PA (PA + En) it was 8.3 %. No mortality was observed in animals treated with 5 pg PA (PA + En). With 150 pg of endotoxin equal protection was observed in 2 and 5 ,ug PA-treated groups (8.3%) which is significantly lower (P~0.05) than the mortality in the untreated group (33.3 96); 1 pg PA did not give any protection in this group (PA + En). With 200 pug of endotoxin, the mortality was 73%. The mortalities with 1, 2 and

LJ Untrratw!

Control

En

PA +En (100&g 1

•m pA

El

61

/x-J En

En(SOfig) PA+En(50mg)

I”

(lOO,ug)

(15Omg)

PA +En

(15Obg)

120 I

NS

PA bg

Fig. 2. Abrogation

of S. r_@irvlurium endotoxin-induced

tein A in mice. Note the significant endotoxin

50 Endotoxin

difference

between

(En) alone (50, 100, 150 fig) as compared

100 /mouse

150

depression

of hepatic aniline hydroxylase

the enzyme

levels estimated

with mice pretreated

after treatment

with protein

by prowith

A (PA) (5 pg).

6 5 pg PA-pretreated groups (PA + En) were 50,46.7 and 40 %, respectively. that all the doses of PA afforded an appreciable amount of protection treated with 200 pg of endotoxin.

This shows in animals

Endotoxin at the 400 ,ug level produced 90% mortality in animals, whereas mortality in mice treated with 1 and 2 pg PA was 80%, which is not significantly different from controls. However, the mortality in animals with 5 pg PA was 70%, which is significantly lower (P < 0.05) than that of controls. Hence, PA at both 2 and 5 yg doses appears to give effective protection against various doses of endotoxin (1 O&400 pug). The highest dose of 5 pg was therefore chosen for all further studies since it produced consistent data. Effect of protein A on hepatic xenobiotic-metabolising enzymes and glutathione levels in endotoxin-intoxicated mice The abrogative effect of protein A on endotoxin-induced depression of hepatic aniline hydroxylase (AH) activity is shown in Figure 2. The endotoxin-treated groups showed a dose-dependent loss of AH activity of 36, 52 and 69% at the 50, 100 and 150 pg doses, respectively, while pretreatment with protein A showed 13, 23 and 40 %

cl

Untreotrd

Control

ml PA Ezi

En (50&g

q El Q III

1

PA+En(5Oag)

NS

ag

lndotoxin

PA+lOONg 15Oug

ondotoxin

PA+15Otig

50

PA

lndotoxin

lOOmu

Endotoxin

100

endotoxin

150

/ mouse

Fig. 3. Abrogation of S. typhimurium endotoxin-induced depression of hepatic aminopyrine demethylase (APD) by protein A in mice. Note the significant (P-C 0.001) difference between APD levels after treatment with endotoxin

(En) alone (50, 100, 150 pg) as compared

with mice pretreated

with protein

A (PA).

protection of enzymatic loss in AH activity in the same group (Fig. 2). No significant difference in enzyme activity was observed in protein-A-treated animals when compared to the controls (Fig. 2). Treatment with protein A alone did not result in any significant elevation of aminopyrine demethylase (APD) activity (Fig. 3). Treatment with endotoxin caused a dosedependent inhibition of APD activity in mice by 26, 57 and 78% in the 50, 100 and 150 fig treated groups, respectively (Fig. 3). However, protein A pretreatment restored APD activity by 18, 32 and 40% in the 50, 100 and 150 pg endotoxin-exposed groups, respectively. The protective effect of protein A on endotoxin-induced depression of glutathione S-transferase (GST) activity is shown in Figure 4. The protein-A-treated group showed no significant change in GST when compared to the control. The 50, 100 and 150 fig endotoxin-treated mice showed a decrease in GST activity of 16, 32 and 45 %>respectively. However, in animals pretreated with protein A there was a protection of 17 and 20% in the 100 and 150 fig endotoxin-treated group, respectively. However, in treated animals, protein A resulted in activity of GST that was almost comparable to that of the control groups (Fig. 4).

El

Untreated

Control

q En (~00~9) PAiEnUOOugf

•n

Protein A

KI

&I

En&Oug)

/?z-J En (15Ofig)

Ll

PA+En (5Otig) E; $ 60 O-7 a

NS

PA+En (15049)

PA Ng Endotoxm

/mouse

Fig. 4. Abrogation of S. ~y~~~uri~ endotoxin-indu~d depression of hepatic glutathione-S-transferase (GST) by protein A in mice. Note the si~ifi~nt difference between the enzyme levels estimated after treatment with endotoxin (En) alone (50, 100,150 pg) as compared to those pretreated with protein A (PA).

,

il

Untrcatrd

i

PA

q

En (50.44

El

NS

Control

Fig. 5. Effect of protein

A challenge

PA+En(

in animals

Protein

A treatment

depletion

50 Endotoxin

glutathione

mice. Note that protein

showing

1 5O~lg)

PA

an hepatic

En(lOOag) PA+En

ag

(En) (50, 100, 1.50 ,~lgf treated

q Q‘I’

(100~~1

En (15Ougl PA+En

Ll

(15O.ag)

lmouso (GSH)

levels in S. f~phi~ur~~~

A (5 p(g) significantly

of GSH levels following

increased

endotoxin

alone did not result in any significant

(P~0.02)

endotoxin GSH levels

inoculation.

increase in GSH content

(Fig. 5). The endotoxin-treated group showed a reduction of 10, 21 and 41% with increasing doses of endotoxin. However, protein A pretreatment restored the GSH content by bringing it back to the normal level in the 50 pug endotoxin group but it was 8 and 14% less than control

in the 100 and 150 pig endotoxin

groups,

respective-

Protein A treatment alone showed a margina increase of 996 in total sulphydryl (-SH) content. Endotoxin treatment caused a 12, 6 and 12% decrease in total SH content in 50, 100 and 150 ,ug exposed animals. In this case protein A pretreatment did not markedly affect the total -SH content (Fig. 6). Efect qf protein A on plasma enzymes, e.g. alkaline phosphatase (ALK-P) , aspartate transaminase ( AST) and alanine transaminase (ALT) , in endotosin-intoxicated mice Protein A treatment alone did not affect the plasma enzyme levels of ALK-P, AST and ALT as compared to the enzyme levels among controls treated with normal saline (Table II). Treatment of mice with endotoxin resulted in a significant elevation of plasma ALK-P (2~70~), AST (3462%) and ALT (103-169%) in a dose-depen-

9

c) ml fzI lzl 120

Untreated PA

El 1,‘.

Ezl

En (5ONg) PA+En(50q)

q

En (?OO~g) PA+En

(lOO&gf

III

En(l50tig)

X

PA+ En ( 150 ug

Q

1

30.98

NS Fig. 6. Effect of protein

Control

A (PA) challenge

PA

100 50 ug Endotoxin /moos8

on hepatic

total sulphydryl

(SH) levels in S. ~yp~~~uri~~ en&-

toxin (En) (50, 100, 150 pg) treated mice.

dent manner. Pretreatment with protein A resulted in substantial lowering of ALK-P (9-21%), AST (I 3-19s) and ALT (19-24s) values (Table II). Although protein A pretreatment did not totally normalise the enhanced plasma enzyme levels caused by endotoxin exposure, it did reduce to some extent the enhanced levels of plasma enzymes. DISCUSSION

The xenobiotic-metabolising enzyme system is responsible for the biotransfo~ation of many drugs, xenobiotics, pesticides and certain endogenous substances including steroids, fatty acids and prostagtandins. The activity of the drug-metabolising enzyme system is necessary for inactivation of many therapeutic agents as well as for the activation of several prodrugs. Toxicity induced by endotoxin, such as early mortality of test animals and enhancement of ALK-P and AST/ALT, has been shown to be significantly abrogated by pretreatment of test mice with protein A. It is well known that endotoxin depresses the xenobiotic-metabolizing enzyme system [24-271, but there is no definite information about their exact mode of action. Endotoxins may act in two or more

10

TABLE

11

EFFECT MICE,

OF VARYING PRETREATED

PHOSPHATASE

DOSES WITH

(ALK-P),

(SO-150 fig) OF ENDOTOXIN

5 .ug PROTEIN

ASPARTATE

(En) CHALLENGE

A (PA) ON PLASMA

TRANSAMINASE

ACTIVITY

GIVEN

TO

OF ALKALINE

(AST) AND ALANINE

TRANSAMIN-

ASE (ALT) ACTIVITIES Treatment

ALK-P

Control

(NS)

Protein

A

alone (5 fig) Endotoxin PA +

(50 pg)

En (50 ,ug)

Endotoxin

fig) PA+En

(s)

AST (GOT)

Increase

(96) ALT (GPT)

Increase

58.3

+ 1.44

0.0

125.28+ 4.2

0.0

45.0 *3.1

0.0

60.6

f 1.83

4.0

127.22k4.5

0.0

45.5

0.0

73.28 & 3.24

26.0

66.9 +5.49 15(9)

167.73f 5.8 136.0 k6.1

34 9(9)

87.7

+3.1

92.41+ 73.6

5.7 *2.85

(%)

103 62(20)

(100

pg) PA+En(lOO~g) Endotoxin

Increase

51.0

188.7

+7.0

51

76.58*4.8

32(13)

164.3

kg.12

31(13)

99.22k6.7

70.0

202.3

k7.7

78.85+5.1

35(21)

174.58k8.94

k3.17

105.8 +6.7 85.7 +3.5

132 88(19)

(150 (15Opg)

Each value is mean

k SE of 6 animals.

PA = 5 pg. Values in parentheses

Activities

are expressed

show percent protection

122.28+5.13 169 92.9 +3.86 155(24)

62 40(14) in pmoles

product

given to respective

formed/min/l

endotoxin-treated

plasma. groups.

ways, first by causing depression or activation of the reticuloendothelial system and secondly by inducing the formation of interferon, both of which appear to be implicated in reducing the hepatic metabolism of drugs. Since cytochrome P-450 is composed of an apoprotein and haem embedded in a lipophilic environment, interaction of the reactive intermediates of either of these components may lead to inactivation. Treatment with endotoxin induces haem oxygenase [28]. which can degrade haem from cytochrome P-450 in hepatic reticuloendothelial cells. Interferon inducers [29] as well as purified preparations of human interferon [30] are known to depress hepatic cytochrome P-450 dependent mono-oxygenase system enzymes. Endotoxin does not affect hepatic drug metabolism in isolated parenchymal cells [31, 321, suggesting a role of certain endogenously released factors. The macrophage, a well-known target for endotoxin, releases a number of secretory products upon endotoxin exposure of which interleukin-1 [33] has been reported to depress liver drug metabolism in endotoxin-treated mice. On the other hand, we have reported recently that protein A stimulates macrophages in both normal and tumourbearing hosts [9]. Our studies have shown that a purified protein (protein A) from S. aureu.s Cowan I can save animals from dying due to toxicity associated with endotoxin (Fig.

11

1). Animals treated with purified protein A did not show any mortality or loss of body weight indicating that protein A itself is not toxic at this dose (data not shown). Pretreatment of animals with protein A, which were subsequently exposed to endotoxin, protects the xenobiotic-metabolizing apparatus from endotoxin toxicity. It may exert its effect by modifying reticuloendothelial function [34], by abrogating this interferon-inducing ability of cells or by inhibiting interleukin-1 producing activity by macrophages, or it may directly act with the endotoxin. Interestingly, we have observed significant effects of protein A in increasing both macrophage numbers and function in normal as well as tumour-bearing hosts [9]. Protein A itself has been reported to have the ability to induce interferon activity [8]. Thus, abrogation of endotoxin activity by protein A could rest& from its competitive binding on similar types of receptors on reticuloendothelial cells as of endotoxin. The latter being too lethal, it exerts its toxicity too rapidly. However, if the endotoxin-binding sites are partly blocked by protein A, lethality can be expected to be reduced. More research, however, is needed to prove or disprove such a hypothesis. One of the known effects of endotoxin on the liver in vivo is hepatocellular necrosis [35]. E. coli endotoxin is known to cause liver injury and thereby an increase in aspartate transaminase (AST) [3.5]. Bacterial endotoxins are also known to be an important cause of cholestasis [36], which increases alkaline phosphatase activity in plasma. The studies mentioned above fully corroborate our results, showing liver injury endotoxin-treated mice and thereby an increase in AST, ALT and alkaline phosphatase activity. Pretreatment with protein A reduces levels of plasma enzymes, thus indicating a lesser magnitude of liver injury. The observations made in this study corroborate our earlier findings with cyclophosphamide [38, 391 and CC4 [40-42]. It has been shown earlier that toxicity induced following exposure to a large dose of cyclophosphamide could be abrogated by protein A pretreatment [38]. Protein-A-pretreated animals showed an excellent recovery, accelerated regeneration of the damage, and repletion of blood cells and enzymes of the hepatic mixed-function oxidase (MFO) system [38]. In conclusion, it may be said that because of the fast recovery of MFO levels this system may provide an excellent model for the investigation of xenobiotic-induced liver damage and its regeneration. Additionally, the present observations appear to indicate a unique interaction between protein A activity and hepatic metabolic activity, thus supporting our previous observations [40]. However, detailed studies are needed to understand the exact mechanism of the action of protein A in protecting the MFO system from the endotoxin toxicity reported here as well as from the cyclophosphamide and CC14 toxicity reported earlier [39,40]. ACKNOWLEDGEMENTS

We are grateful to Dr. Mukul Das and Dr. B.M. Gupta for critical evaluation of the manuscript and for their fruitful suggestions. Thanks are also due to Mr. Chedi

12

La1 for sustained help and to Mr. L.K. Goswami for secretarial assistance. Financial assistance to one of the authors (P.D.D.) from the Council of Scientific and Industrial Research (CSIR), India, is gratefully acknowledged. REFERENCES 1 Heikkill,

J.J. and Brown,

by bacterial

pyrogen.

2 Sakaguchi,

S.. Sakaguchi.

with endotoxin. 3 Green.

I.R. (1977) Hyperthermia

0. and Hus, C.C. (1979) Metabolic

disorders

induced

of lipids in mice administered

K. and Kasai,

N. (1979) Endotoxin

glycohpid

as a potent

Immunol.

23.89-94.

B.R., Yaffe, S.J. and Witmer,

ma1 drug metabolism and

Abdul.

biochemical

depressor

of the hepatic

C.M. (1982) Effects of endotoxin

in vivo and in vitro. Xenobiotica

T.. Rahman

cardiographic

of mouse

Inst. 63.407-502.

bolizing enzyme systems in mice. Microbial.

6 Mohammed,

polysomes

A. (1979) Corynebacterium parvum effects on the biochemistry

S. and Dorbjansky.

5 Sanewane,

of brain

Jpn J. Med. Sci. Biol. 32.95-98.

serum and liver. J. Natl. Cancer 4 Egawa,

and disaggregation

Life Sci. 25,347-352.

M. and Parmer, changes

upon rat hepatic

microso-

12.3033312.

N.S. (1986) Decrease

following

drug meta-

endotoxin

by naloxone

induced

shock

of some electro-

in rats.

Toxicon

24,

101-103. 7 Newton,

R.C. (1986) Human

interleukin-I 8 Catalona.

secretion

monocyte

9 Prasad,

killing and ADCC.

A.K.,

Singh,

activity in protein

K.P..

following 49704974.

Saxena,

A treated

IO Ray, P.K., Rayc~dudhuri, plasma

anti-tumor

12 Ray, P.K., Bandyopadhyay.

A containing

induced

Immunopharmacol.

A containing

SK. (1983) Inhibition agent. Immunol. S., Dohadwala.

by S. aureu.r protein

of

A aug-

of mammary

9, %-%I.

adenocarcinomas

in rats

~~u~h.~~oc~c~usaweus. Cancer

Res. 42,

tumor growth

by purified pro-

12,4533464.

M., Canchanapan,

A. Cancer

macrophage

Immunotoxicol.

of rat mammary

Commun. Immunol.

D., Allen. P., Bandyopadhyay,

Jr., J.E., Basset. J.J. and Cooper,

of the induction

N. and Ray, P.K. (1987) Increased

animals.

protein

doses of protein

13 Ray. P.K., McLaughlin,

Mathur,

S. and Allen, P. (1982) Mechanism

11 Ray. P.K. and Bandyopadhyay,

activity with nontoxic

R.E. (1981) Interferon

A.K.,

over

parameters

Biol. 39.299-312.

291, 77-79.

tumor regressed

adsorption

tein A: a potential

Nature

of interleukin-I:

J. Leukocyte,

W.J.. Ratliff, T.L. and McCool,

ments natural

protein

production

by lipopolysaccharide.

P. and Mobini,

Immunother.

S., Idiculla,

D.R. (1984) Adsorption

A.. Clarke,

of plasma

non viable Staphylococcus aweus Cowan

J. (1984) Antitumor

18, 29-34. L., Mark,

from tumor

R., Rhoads

bearing

I: possible mechanism

host over

of antitumor

re-

action. J. Biol. Res. Modif. 3, 293-302. 14 Ray, P.K., McLaughlin, Cooper.

D., Mohammed,

J.. Idiculla.

D.R. (1981) Ex vivo immunoadsorption

treatment.

In: B. Serroui

cer Patients.

and C. Rosenfeld

~Isevier~North-Holland

(Eds.), Inlmune

Biomedical

16 Cochin,

J. and Axelrod.

ic administration 17 Kato,

in fibrosarcoma

nalorphine

W.H.. Pabst, M.J. and Jakoby,

and correlation

and pharmacological

and normorphine.

J.R. (1965) Effect of starvation

somes of male and female rats. J. Pharmacol. 18 Habig,

Complexes

R., Basset, J.G. and

~ a new modality

and Plasma

of cancer

Exchanges

in Can-

of plasma

immunoglobulins

with

mice. Ind. J. Exp. Biol. 25.514518.

J. (1959) Biochemical

of morphine,

R. and Gillette,

bearing

Jr., J.E., Mark,

Press, pp. 197-207.

15 Saha, S.. Prdsad, A.K. and Ray, P.K. (1987) Changes tumor growth

A., Rhoads

of IgG or its complexes

changes

J. Pharmacol.

of NADPH

in the rat following

chron-

Exp. Ther. 125. 105-l 10.

dependent

enzymes

in liver micro-

Exp. Ther. 150, 2799284.

W.B. (1974) Glutathione-S-transferase:

the first enzymatic

step

in mercapturic acid formation. J. Biol. Chem. 249, 713~-7139. 19 Jollow. D.J.. Mitchell. J.R., Zampaglione, N. and Gillette, J.R. (1974) Bromobenzene induced liver necrcsis: protective role of glutathione and evidence for 3,4_bromobenzene oxide as the hepatotoxic intermediate.

Pharmacology

11,151-169.

13

20 Reitman,

S. and Frankel,

oxalacetic

and glutamic

21 Wotten.

S. (1957) A eolorimetric

pyruvate

transaminases.

I.D.P. (1964) In: Microanalysis

method

for the dete~ination

Am. J. Clin. Pathol.

in Medical

Biochemistry,

of serum glutamic

28, 5663. 4th edn. J.A. Churchill

Ltd., Lon-

don, pa 101. 22 Lowry,

O.H..

Rosebrough.

Folin phenol reagent. 23 Finney.

N.J., Farr,

J. Biol. Chem.

D.J. (1978) In: Statistical

Ltd., London

A.L. and Randall,

Methods

and High Wycombe,

R.J. (1951) Protein

measurement

with the

193,265-275. in Biological

Assay, 3rd edn., Charles

Griffin and Company

pp. 3722385.

24 Gorodisher, R.. Krasner. J.. McDevitt, J.J., Nolan, J.P. and Yaffe, S.J. (1976) Hepatic microsomal drug metabolism after administration of endotoxin in rats. Biochem. Pharmacol. 25, 351-352. 25 Abernathy,

C.O. and Zimmerman,

lism in mice. Res. Commun. 26 Williams,

J.F. and Szentivanyi,

the endotoxin

alteration

ticoid-induced 27 Williams,

H.J. (1980) Effect of endotoxin

Chem. Pathol.

29. 1933196.

A. (1973) Investigation

of adrenergic

of hepatic heme oxygenase,

tryptophan

oxygenase

activities.

J.F.. Low&t, S. and Szentivanyi,

oxidase system in C3H/HeJ 28 Gemesa.

D., Woo. C.H.,

macrophage 29 Renton,

Fudenberg,

microsomal

and prosta~andin

Immunopha~acology

inff~~ences in

oxidase and glucocor-

675-86. depression

mice. Immunopharmacology

H. and Schmide,

and liver by endotoxin.

on in vivo drug metabo-

mixed function

A. (1980) Endotoxin

and C3H/HeN

K.W. and Mennering.

xygenase

tolerance

Pharmacol.

of hepatic

mixed function

2,2855291.

R. (1974) Stimulation

of heme oxygenase

in

J. Clin. Invest. $3. 647651.

G.J. (1976) Depression

system with administrated

interferon

of hepatic cytochrome

inducing

agent.

Biochem.

P-450 dependent

Biophys.

mono-

Res. Commun.

73,

343-348. 30 Single, G.. Renton, drug metabolizing

K.W. and Stebbing, systems.

31 Berry, L.J. (1977) Bacterial

33 Ghezzi,

P.. Saccardo,

34 Singh,

K.P.,

of protein

of liver drug metabolism

A.K.,

Prasad,

A on mast cell numbers

P-450

on glycogenolysis,

glu-

Proc. Sot. Exp. Biol. Med. I55.216218.

B., Villa. P., Rossi, U., Bianchi,

Saxena.

cytochrome

5239318.

B.J. (1977) In vivo vs. in vitro effects of endotoxin

and glucose utilization.

leukin 1 in the depression

on hepatic

23, 283-287.

toxins. CRC Crit. Rev. Toxicol.

32 Filkins, J.P. and Buchanan, coneogenesis

N. (1981) Effect of interferon

Pharmacologist

A.K..

M. and Dinerello,

by endotoxin.

Dwivedi,

and macrophage

P.D., Zaidi, phagocytic

CA.

(1986) Role of inter-

Infect. Immun.

54,837-840.

S.I.A. and Ray,

activity.

P.K. (1987) Effect

Immunopharmacol.

Immuno-

toxicol. 283, 281-~298. 35 Lee, D.W., Kim, C.S.. Lee. Y .B. and Kim, D.S. (1973) A study of hepatic

injury induced

by endotoxin

in rats. Yonsei Med. J. 19, 19. 36 Curtis,

M.J., Jenkins,

hormone

on plasma

37 Minuk,

G.Y.,

Rascanin,

effects of bacterial 38 Ray, P.K.,

H.G. and Butler, enzyme activities N., Sarjeant.

products

Dohad~dla,

function

M., Bandyopadhyay,

uptake SK.

toxicity using protein

by isolated

system

endotoxin

and adenocortical

of cyclophosphamide

rat hepatocytes.

and McLaughlin, A. Cancer

M. and Ray, P.K. (1985) In vivo protection

oxygenase

coli

fowl. Res. Vet. Sci. 28,445O.

E.S. and Pai, C.H. (1986) Sepsis and cholestasis:

in C-14 teusocholete

large dose cyclophosphamide 39 Dohadwala.

E.J. (1980) The effect of E.

in the domestic

D. (1985) Rescue

Chemother.

by protein

treated

rats.

the in vitro

Liver 6, 199-204.

Pharmacol.

A of hepatic

Cancer

of rats from

14,59-62.

microsomal

Chemother.

mixed

Pharmacol.

14,

135-138. 40 Srivastava protein

S.P., Singh.

A of hepatic

Pharmacol. 41 Singh,

K.P., Saxena,

microsomal

A.K.,

Seth, P.K. and Ray, P.K. (1987) In vivo protection

mixed function

oxidase

system of CCL administered

36.405554058.

K.P., Saxena,

A.K.,

(1988) Protection against Toxicol. 8, 407410.

Zaidi, carbon

S.I.A.,

Dwivedi.

tetrachloride

P.D., Srivastava,

induced

hepatotoxicity

S.P., Seth, P.K. and Ray, P.K. in rats by protein

42 Singh, K.P., Saxena. A.K.. Srivastava, S.P., Seth. P.K., Zaidi, S.I.A., Dwivedi, (1988) Protection against carbon tetrachloride induced lymphoid organotoxicity Toxicol.

by

rats. Biochem.

Lett. (submitt~).

A. J. Appl.

P.D. and Ray, PK. in rats by protein A.