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St. Johnrs

studies of the Pancreatotoxioity of l-~ydn0-2-hydr0~y-3-butene

A thesis submitted to the School of Graduate Studies in partial fulfillment of the requirements for the degree of

Master of Science

Toxicology

Memorial University of Newfoundland

October 1991 Newfoundland

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SauuosMd sap UO!I!SO~S!P el e asam eue3

BP SBlEldUa~e S p BPJUaW JnOd l p s 03 enb '9~081ed pelsemg! 01 awl01 anblenb snos la a~a!uw enblanb ap e W s l l e n e s l s e l n a u r B u ~ ' l e ~ ~ ~ u u 0 1 A y ~ oS?qlm ap seldrn SOP e,puan no renqulstp u s m w Am 6q s s q 1 mq/sq lo send03

ralCd 'a,tnpo>dar ep e ~ e v e ~ np aleweu las loewulsp 'q.a?npaxla~ 01 epm3jo ellb?~Iayq*l 31 8 lueueuuad mlsnpxa uou m n w 8 l m an Bulmof~c e x a l l anlsnnva (a a!qmA?u! e ? u w aun p m e a Jna8ne.i .uou * q m n a i im pa)uk;fi seq ;owba

'eqL

A raceaio mixture of (R)- and (S)-I-cyano-2-hydroxy4- butene (CHB) war synthesized using 1.3-butadiene as a starting compound. he effects oL CHB on pancreatic non- protein thiol and glutathione S-transferase activity were investigated in adult male rats when administered orally in olive oil at a dose of 200 ng/kg. At 4 hours after dosing, pancreatio non-protein thiol content was depleted but rebounded at 24 hours t o 1 times oontrol values. At 120 hours aiter dosing, pancreatic non-protein thiol levels were still elevated over control levels. Hepatic non-protein thiol levels were greater than control levels at 4 hours after dosing, but were not significantly different from control values at 24 and 120 hours after dosing. The pattern of pancreatic non-protein thiol changes with synthetic CHB resembles that reported with the natural 1s)-

~ S O ~ B T Of CHB.

Glutathione S-transferase activity war decreased at 4 hours Eollowing CHB treatment in the rat pancreas, and elevated at 24 hours. Hepatic glutathione S-tranrferase

iii activity was not altered by CHB treatment.

The effect of inoreasing intraduodenal doses of CHB (25-200 mglkg) on rat pancreatic juice secretion and bile secretion was investigated in conscious rats. CHB was administered intraduodanally and caused a stimulation in pancreatic juice secretion followed by a return to control values at the lower doses. At higher doses, CHB causes the same early seimulstion, but is fallowed by a depression of pancreatic juice flow. 1\11 doses of CHB administered resulted in a dose-dependent decrease of protein concentration in panorastic juice.

ctlB caused the pancreeta of treated rats to beoone oedenatous, a condition also seen in certain forms of acute panoreatitis. The severity of the oedema was dose-dependant and oould be visually detected. The exudate collected Eram these pancreata had protein concentrations comparable to plasma.

Radioactive CHB (["C]) was synthesized and administered to rats intraduodenally that were also fitted with biliary fistulae. CHB was found to be excreted over 8 hours in the bile and urine, with higher amounts excreted in the urine.

Little ["c] was detected in the pancreas and kidneys, but did accumulate in the adipose tissue (7.7%) and in the liver

(5.5%).

These results indicate that synthetic CHB is a powerful and selective pancreatotovin and the present data are not

iv

incompatible with a glutathione-mediated mechanism, as is known to uccur with other unsaturated nitriies.

ley words

l-~yan0-2-hyd~-0xy-3-butene POnCreDB

l i v e r d e t o x i c a t i o n qlutathione

q l u t a t h i o n e S-transeeraee n i t r i l e

p r o t e i n bile m e t a b o l i t e s

I would l i k e t o thank t h e l a t e D r . James A . warrowman f o r g i v i n g m e t h e o p p o r t u n i t y t o p a r t i c i p a t e i n t h i s research, and f o r h i s guidance t h r o u g h o u t .

I owe a g r e a t d e b t t o M r . G . chernenko, for h i s p a t i e n c e i n t e a c h i n g me l a b o r a t o r y t e c h n i q u e s and computer b a s i c s .

MYs u p e r v i s o r y committee of D r . K. Keough, D r . J. O r r , a n d D r . A. Rahimtula were e x t r e m e l y h e l p f u l i n t h e i r a d v i o e a n d s u g q a r t i o n r throughout t h e p r o j e c t .

I g r a t e f u l l y acknowledge t h e f i n a n c i a l a s s i s t a n c e p r o v i d e d t o ma by t h e School of Graduate S t u d i e s and t h e F a c u l t y of Medicine.

P i n a l l y , I would l i k e t o e x p r e s s my t h a n k s t o George and A n t j e Maher, my p a r e n t s , f o r t h e i r c o n s t a n t encouragement and f a i t h i n me.

able ef contents page

1. Introduction 1

1.1. G1~009in0111te6 1

1.1.1. Beneficial Effect of Crucireraur

Diets 4

1.1.2. G l ~ ~ o ~ i n o l a t e Breakdown Products 7 1.1.3. Nitriles from Crucifers 9 1.2. Metabolism of Xenobiotics 15

1.2.1. Glutathione and Glutathione S-

Transfarase 17

1.2.2. Mercapturic Acid Fornation 21 1.3. P a n c r e a t o t o ~ i n ~ 22 1.4. Objectives of the present study 26

2 . Methods and Materials 27

2.1. Synthesis of CHB 27

2.1.1. Synthesis of 5-~inyl-2-isoxazoline 27 2.1.2. Synthe-is of CHB from 5-vinyl-2-

isarazolina 30

2.2. on-Protein Thiol and Glutathione S-

Transferase Induction 31

2.2.1. Animals 31

2.2.2. Treatment with CHB 31

2.2.3. Cell Fractionation and Protein

Precipitation 32

2.2.4. Principle of Protein Assay 34 2.2.4.1. Preparation of Dye Reagent 34 2.2.4.2. Protein Standard 34 2.2.4.3. Protein Array Method 35 2.2.5. Principle of Nan-Protein Thiol

Assay 35

2.2.5.1. Standard Preparation 38 2.2.5.2. Determination of NP-SH Groups

in samples 38

2.2.6. Principle of GSH-T Aotivity

Assay 39

2.2.6.1. GSH-T Activity Assay Method 39

Table of Contents foontinued) page 2.3. me Effact of cHB on Pancreatic Juice

and Bile Secretion

2.3.1. A n i m a l s 4 3

2.3.2. Surgery 43

2.3.2.1. Anaesthesia 43

7.3.2.2. Bile Duct cannulation 4 3 2.3.2.3. Duodenal Cannulation 4 3

2.3.2.4. Closure 4 4

2.3.2.5. Post-operative Housing and 4 9 Sacrifice

2.3.3. Test DosarJes and Administration 49 2.3.4. Pancreatic Juice and Bile Sample ⁴ 9

Collections

2.3.5. Determination of Protein 50 Concentration in Pancreatic Juice 50 2.3.6. Principle of Bile Acid Concentration

Assay

2.3.6.1. Preparation o f Reagents 51 2.3.6.2. Assay Procedure 51 52 2.3.6.3. Calculations

2.3.7. Datemination of Pancreatic water 53 Content and Protein Concentration of

Pancreatic Exudate 53

2.4. Distribution of Radioactive CHB in the Rat 54 2.4.1. Animal6

2.4.2. synthesis of ["C] CHB 54

2.1.3. Surmerv 54

,

-.

2.4.4. sacrifice . .

54 2.4.5. Radioactive Test Meal Canposition 55 2.4.6. Test Meal Administration 55 2.4.7. Sample Collection

2.4.8. Tissue Samples 56

56 2.4.9. Liquid Scintillation Counting

2.5. Statistical Methods 57

3 . Reaulta 57

58 3.1. Effect o f a Synthetic, Raceaic Mixture ^{o f}

1s)- and ( R ) - CHB on NP-SH Levels and GSH-T Activity in the Pancreas and Liver

of the Rat 58

3.1.1. Pancreatic and Hepatic NP-SH

3.1.2. Pancreatic and Hepatic GSH-T 58 Activity

3.2. E f P B E t Of Increasing Doaea oC CHB an 59 1)ancreatic: Juice secretion and Protein

concentration 6 8

3.2.1. Pancreatic Juice Secretion 6 8

Table of Contents Icontinuedl 3.2.2. Protein Concentration of

Panoreatio Jvice

3.3. Effect of Increasing Doses of CHB on Bile Secretion and Bile Acid Concentration 3.3.1. Bile secretion

3.3.2. Bile Acid Concentration 3.4. Pancreatic Water Content 3.5. Disposition of Oral CUB in the Rat

3.5.1. Radioactivity in the Bile and Urine

3.5.2. Radioaotivity in the Liver, Pancreas, Kidney, and Adipose Tissue

I . D ~ S E Y S S ~ O ~

4.1. Effect of CHB on NP-SH and GSH-T in the Liver and Pancreas

4.2. GSH and Nitrile Metabolism 4.3. Effects of CHB on Pancreatic Juice

Secretion and Protein Concentration 4.4. Effect of CHB on Bile Secretion and Bile

Acid Concentration

4.5. Pancreatic Oedeaa in CHB treated Animals 4.6. Disposition of CHB

conslusions Refermnoes

Glucosinolates and their breakdowr. 3 p r o d ~ o t s

CUB and CHEB 11

Glutathione 19

Mercapturic Acid Formation 24

Synthesis of CHB 29

The reaction of DTNB with a sulfhydryl

group t o form 2-nitro-5-nercaptobenzoic acid 37 The reaction of CDNB with GSH to Para a

glutathione oonjugate 41

Bile Duct Cannulation 46

Duodenal Cannulation 48

Effect of CHB on PrP-SH content in rat pancreas 61

~ f f e c t of CHB on NP-SH contents of rat liver 63

~ f f a c t of CHB on GSH-II activity in rat panoreae 65 Effect of CHB on GSH-T activity in rat liver 67 Effect of olive oil on rat pancreatic

juice secretion 71

Effect of 2 5 mg/kg CHB on rat pancreatic

juice eecretion 73

~ f f e c t of 50 mg/kg cnB on rat pancreatic

juice secretion 75

~ f f a c t of 100 nglkg CUB on rat pancreatic

juice secretion 77

~ f f e c t of 150 mg/kg cHB on rat pancreatic

juice secretion 79

Effect of 200 mg/kg CHB on rat pancreatic

juice secretion 81

Effect of increasing doses of CHB on rat pancreatic juice secretion with time 84

~ f f e c t of olive oil en protein concentration

of rat pancreatic juice 86

Effect of 25 mgtkg CHB on protein

concentration of rat panoreatic juioe 8 8 Effect of 50 mg/kg CHB on protein

concentration of rat panareatic juice 91 Effect of 100 ng/kg CHB on protein

concentration of rat pancreatic juice 93

xi

List or Figures (continued]

Effect of 150 nglkg CHB on protein concentration of rat pancreatic juice Effect of 200 aglkg CHB on protein concentration of rat pancreatic juice Effect of increasing doses of CHB on protein concentration of rat pancreatic juice with tine

Effect of olive oil on bile secrrtion in the rat

Effect of 25 mglkg CHB on bile secretion in the rat

Effeot of 50 aglkg CHB on bile secretion in the rat

Effect of 100 ngjkg CUB on bile secretion in the rat

Effect of 150 nglkg CHB on bile secretion in the rat

Effect of 200 ngjkg CHB on bile searetian in the rat

Effect of increasing doses of CUB on rat bile secretion with time

Effect of 200 ngjkg CHB on bile acid conclntration in rat bile EPfect of inoreasing doses of CUB on pancreatic water content

Pancreata from control animals and from treated animals

Radioactivity recovered in rat bile for 8 hours after dosing with I"C1 CHB Cumulative radioactivity recovered in bile and urine for 8 hours after ["c] CUB dosing Radioactivity recovered in rat organs 8 hour6 after dosing

Page

CHB OZT SCN' CHBB GSH GSH-T GSSG NP-SH DTNB

1-cyana-2-hydroxy-3-butenn oxazoline-2-thione thiocyanate

l-cyano-2-hydroxy-3,4-epithiobutane reduced glutathione

glutathione S-transferase oxidized glutathione non-protein thiol

5 ~ , 5 r - d i t h i o b i s - ( 2 - n i t r ~ b e n ~ ~ i ~ acid)

1-chlaro-2.4-dinitrobenzene

Dedication

This thesis is dedicated t o the memory of Dr. James A. Barroman

and

Chapter 1 Introduetien

1.1. Gluc0sin01ates

olucosinolstar a r e compounds whose breakdown products are responsible for the pungent flavouring of cultivated plants such as turnip and cabbage (Fenwick et al, 1983).

Most glucosinolate-containing plants belong to the C r u c i f e r a e family, many of which are major co~nmercial crops.

A variety of crucifere are used as pasture, forage, and Silage for 1iveStOck and belong to the genus B r a s s i c a (Tookey et a l , 1980). Mustard, broccoli, cabbage, cauliflower, turnip, and brussel sprouts are all commonly consumed Brassica plants. It is interesting to note that ancient cultivation of B r a s s i c a crops was performed to use the plants tor aedioines (Fenwick et a l , 1983).

Gluoorinolater exist as anionic salts in plants usually associated with potassium (Figure 1-1). They occur in all parts of the plant. An aocompanying enzyme system is present which can hydrolyse glucosinolates, but is

;.hyrically separated from the glucosinolate itself in the

Figure I-lr Qluoosinolstes and their' breakdown products

/S-C6H,,0~ H 2 0 + D - Glucose R-C

U N 0 S o j Thioglucosidase PLANT

' [.-a] ⁺

Glucoslnolale R - N = C = S

Isothiocyanate 3 - S - C s N R - C s N

Nilrile

+

-

4

plant. ÿ he enzyme w a r called myrosin or nyrorinane (Daxenbichler et a l , 1966) in early investigations, but the recommended name is thioglucoside hydrolase (EC 3.2.3.1.) (Tookey et al, 1980). This thioglucosidare has haen identified in the leaf, stem, and roots of plants oontaining gluconinolates (Fenwick et al, 1983).

Thioglucosidasa hydrolyres gluco=inalates whenever the plant containing them is wet and orushed. The products of this hydrolysis ere glucose, bisulfate ion, and an organic component (Figure 1-1). This remaining organic portion usually forms a nitrile with the 1058 of sulfur, or intra- molecular rearrangement may occur yielding thiocyanates and isothiocyanates (VanEtten et el. 1969a). Another rearrangement may result in the formation of cyolic compounds such as oxazoline-2-thiones (OZT) and epithionitriles. Which aglucon product is formed depends on factors such as pH, the presence of Fez' ions, and the presence of an epithiospecifier protein (Tookey et el, 1980). The R- group of the glucosinolate itself nay oontain a cyclic organic moiety, as is found in indole

gluconinolates.

1.1.1 Benefioial BffeOt of cntciferous Diets

Early work by Wattenberg (1971) showed that cabbage, broccoli, and brussel sprouts added to rat diet caused a significant increase in the activity of benzo[e]pyrene

5

hydroxylasa in rat int~stine. This enzyme and other aryl hydroxylasas are Phase I metabolizing enzymes which t r a n s f o m aryl hydrocarbons into more water soluble oompounds, by a manoxydation reaction that insects a n aton of oxygen between a carbon and a hydrogen. Further work in this area by M u b et el (1975) shoved that the inducing chemicals in these Vegetables ware glucosinolate derivatives, indole-3-carbinal, indnle-3-acetonitrile. and 3*,3,-diinodoylmethane. the most potent of these being indole-3-carbinol. These three derivatives induced benzo[a]pyrene hy4rarylase activity in the liver and small intestine. This work was supported by the studies of Pantuck st a1 (1976). The same three glucosinolate derivatives tested were f w d to increase the intestinal metabolism of four xenobiotics (hexobarbital, phenacetin, 7 - ethoxycoumarin, and benzolalpyrene) with indole-3-carbinol again being the most powerful inducer.

cruaiferous diets are known t o decrease the incidence of colon and rectal cancer. Graham et a1 (1978) reported that the risk of colon cancer in males end females deoreases with amount of cabbage, brussel sprouts, and brocooli consumed. These are all cruciferous vegetables containing qlucosinolater.

stoewsand et el (1978) found that feeding oauli€louer t o rats enhanced hepatic xenobiotic metabolizing enzymes.

he cauliflower diet also resulted in reduction of fatty

6

livers produced by dietary polybrominated biphenyls (PBB) and reduced the toxic effects of aflatoxin 8,. Both PBB's and aflataxin 8, are well known carcinogens.

Glutathione S-transferaaea (GsH transferases) in the liver were found to be induced by feeding weanling rats brussel sprouts (Godlewski et el, 1985). Bath a glucosinolate-containing extract from brusael sprouts and the remaining residue (not containing glucosinolates) also induced GSH transferases. This information suggested that Other compounds besides glucosinolates in cruciferous vegetables may play a role in soma datoxication mechanisms.

Diets supplemented with cauliflower, kale, and brussel sprouts given to mice were found to induce the activity of ethoxycouearin O-deethylase activity in the mouse liver

(Bradfield et a l , 1 9 8 4 ) . This enzyme is responsible for the deethylation of 7-ethoryooumarin, a powerful carcinogen, by making it nore water soluble and hence more readily excreted. Cauliflower and brussel sprouts dicts did not induce the activity of mouse hepatic aeyl hydrocarbon hydroxylare, but did induce the activity of mouse hepatic GSH transferases. The authors pointed out that increases in enzymic activity also oomr as s response to toxic stress, and that the effective compounds in the vegetables may be toxins themselves.

TWO kinds of cabbage (White and Savoy-type) fed to rate in 25% of the diet were found to significantly increase

7

d e e t h y l a t i o n of e t h o x y r e s o r u f i n (ERR: a c a r c i n o g e n , a o a n n e l l e t a 1 (1988).

1.1.2. Q l u c o s i n o l a t e Breakdown Produota AS p r e v i o u s l y mentioned, many c r u c i f e r s a r e of i m p o r t a n t economic v a l u e . o n e of t h e s e is r a p e s e e d , which i s d e r i v e d Erom v a r i e t i e s of B r a s s i c a napus and ~ r a s s i c a c a m p e s t r i s . O i l i s e x t r a c t e d from t h e s e e d and t h e remaining meal i s u s e d t o Eaed l i v e s t o c k . However, it c a n o n l y be f e d i n l i m i t e d amounts because it c o n t a i n s t o x i c s u b s t a n c e s as d e s c r i b e d below (Tookey e t e l , 1980).

Rapeseed meals t r a a t e d t o y i e l d products h i g h i n n i t r i l e a or g o i t r i n ((SJ-5-vinyl-OZT) cause decreased weight g a i n i n r a t s and c h i c k s ( S r i v a s t e v a e t a l , 1 9 7 5 ) . R a t s are more s e n s i t i v e t o t h e n i t r i l e - r i c h d i e t t h a n c h i c k s , end have p a l e e n l a r g e d kidneys. Turkeys fed r a p e s e e d p r o d u c t s f o r 1 6 wb-ks developed s c l e r o s i s of t h e l i v e r showing m u l t i p l e f o c a l a n d l o r s o l i t a r y h e p a t o c y t i c necroses p r e c e d i n g f i b r o s i s o f t h e l i v e r (Unenuea st a l , 1977). The a u t h o r s proposed t h a t g l u c o s i n o l s t e s and t h e i r h y d r o l y t i c p r o d u o t s a r e t h e l i k e l y cause f o r t h e d i s e a s e d l i v e r s . Martland and B u t l e r (1984) found t h a t f e e d i n g r a p e s e e d meal t o l a y i n g h e n s r e s u l t e d i n l i v e r hemorrhage, r e t i c u l o l y s i s , t h y r o i d enlargement, and o t h e r biochemical changes. Both h i g h and low g l u c o s i n o l a t e c o n t a i n i n g meals t e s t e d c a u s e d t h e s e problems.

8

Goitrin, found in rapeseed, was found to be thyroid- suppressing. Gaitrin and its enantiomer (R)-5-vinyl-OZT are both antithyroid agents derived from glucorinolates having a side chain of (R)- and (SI-2-hydroxy-3-butena. These cornpounds act by inhibiting the uptake of iodine by the thyroid and by interfering with thyroxine secretion (Astwood st al, 1949).

Thiocyanate ions (SCK), also derived from glucosInolate hydrolysis, are antithyroid agents as well.

They prevent the accunulation of iodine by the thyroid by inhibiting uptake and by causing the discharge of iodine from the gland (VanderLaan and Vanderlaan, 1947).

I ~ o t h i o ~ y a n e t a s also cause changes in thyroid function.

Langer and Stalc (1363) found that allylisothiocyanste, a natural mustard oil component, significantly depressed radioiodine uptake by the rat thyroid. It was later determined that metabolic detovication of isothiocyanates, such as allylieothiocyanate, involves the release of the thiooyanate ion from the molecule which is believed t o be responsible for the thyroid inhibition of these ~DTnpoundr (Wood, 1975).

A substantial body of literature describes the deleterious effects oE glucorinolates given in animal feeds including the important antithyroid effects of OZT conpounds, thiocyanates, and isothiocyanstea (Tookey et a l , 1980; enw wick et a l , 1983).

9 1.1.3. Nitrilms from Crucifers

Nitriles obtained fron ccuciferous plants have also been investigated with respect to toxic effects. ~byssinian kale (Crambe sbyrsinica) contains a high level of (s)-2- hydroxy-3-butenyl glucosinolate at 80-97 mglgm of plant

(Daxenbichler et a l , 1965). A mixed nitrile extract obtained from C r a m s seed and fed t o rats caused death at 0.2% by weiqht of the diet. At 0.1% of the total diet, rats had decreased weight gain along with liver and kidney lesions (VanEtten e t al, 196913). Daxenbichler st el (1968) previously showed that the mixed nitrile extract fron crambe leal contained 2S-l-cyano-2-hydroxy-3,4-epithiobutanes

(erythro- and threo- forms), abbreviated to CHEB, and (S)-1- cyano-2-hydroxy-3-butene, abbreviated t o CllB (Figure 1-21, Both of these compounds hava been investigated for target Organ toxicity and mode of action.

Niehie end Daxenbichler (1980) investigated the teratogenicity of glucasinolates and some at their derivatives including CHEB and CHB. Both compounds were administered daily to rats starting on day 8-9 of gestation by subcutaneous injection (s.o), and continued until 11-12 days after the beginning of treatment. Neither Was found t o be teratogenic, but at the highest doses tested (95 mg/Kg body weight for CHEB, 150 mglkg for CHB) both caused significant decreases in fetal weight (sc dose). CHB also resulted in a significant increase in _occurence of resorbed

r i p r e 1-2: I-cyano-bydroxy-3-buten- (CAB) (I) and

1-oylmo-2-hydrorry-3,4-epitbioh~tane (CBBB) (2)

12

fetuses. Both compounds caused depressed weight gains 1s.C.

dose) and increased adrenal weights in pregnant rats (s.c.

dose). pregnant rats treated with CHB had decreased liver weights and hepatic necrosis (s.0. dose of 175 mglkg). CHEB was nephratoxic to both pregnant female and male rats causing increased kidney weights (=.a. dose of 95 mqlkg).

The effect of feeding diets containing 75, 150, and 300 mglkg CHEB to male rats war examined by Gould et a1 (1980).

All diets caused decreased weight gain in the animals.

Livers Of CHEB treated animals had dose-related bile-duct hyparplasia and fibrosis. A11 rats fed the highest dose had severe liver damage and nephrocytomegaly. Three rats out of six fed the highest dose of CHEB had pancreatic acinar cell karyomegaly.

The nephrotoxicity of CHEB was further exarnintd by Gould et a1 (1985). Rats gavaged wit" CHEB had increased serum urea nitrogen and creatinine and decreased glanerular filtration rates. Histology of Kidneys showed necrosis and coarse vacualation of proui~ull tubular epithelial cells.

The pars recta (4 segment) of the proximal tuaule was the initial target of CHEB. More extensive lesions were observed with larger doses and/or l m g e r times of CHEB treatment.

The damage to rat organs caused by CHEB is discussed here as CHEB is similar in structure to CHB, the compound investigated in the present work. Nallig et a1 (1988)

13

s t u d i e d t h e a f f e c t of n a t u r a l (S)-CUB p r e p a r e d from crambe e b y r s i n i c a o n r a t s by gavaging t h e n w i t h 200 nqlkg CUB.

They found upon examining organs by l i g h t nicrosoopy t h a t o n l y t h e p a n c r e a s of t r e a t e d animals was damaged. Some PanCreata o f t r e a t e d animals were e n l a r g e d and oedemstous a f t e r 2 days. seen i n a l l pancreata of t r e a t e d r a t s were decreased zymoqen g r a n u l e s and f r a q m e n t a t i o n of a c i n a r c e l l s . The a u t h o r s e t a t e d t h a t t h e s e and o t h e r changes seen i n t h e p a n c r e a t a of CUB t r e a t e d r a t s were i n d i c a t i v e of both n e c r o s i s and a p o p t o s i s (programmed c e l l d e a t h ) . Urinary t h i o c y a n a t e e x c r e t i o n was monitored t o d e t e r n i n e i f cyanide r e l e a s e from CHB war t h e cause of t h e p a n c r e a t i c damage observed. Cyanide is d e t o x i f i e d i n t h e l i v e r by t h e enzyme rhodanase t o y i e l d t h i o c y a n a t e , which is much l e s s t o x i c t h a n cyanide ( S i p e s and G a n d o l f i , 1986). ~ e s u l t s showed t h a t low molar amounts of t h i o c y a n a t e were e x o r e t e d i n propo=tion to t h e molar dose of CHB a d m i n i s t e r e d and n o t enough t o cause p a n c r e a t i c l e s i o n s which i n any case a r e not a f e a t u r e of c y a n i d e t o x i c i t y . These r e s u l t s show t h a t n a t u r a l CHB has a s e l e c t i v e p a n c r e a t o t o x i c e f f e c t i n r a t s e i t h e r d i r e c t l y or as some m e t a b o l i t e o t h e r t h a n cyanide.

F u r t h e r worK by W a l l i g and J e f f e r y (1990) showed t h a t a s i n g l e o r a l dose of CUB (200 ng/kg) reduced g l u t a t h i o n e (CSH) c o n t e n t i n t h e pancraar of r a t s u n t i l s i x hours a f t e r dosing. A f t e r t h i s t i m e CSH c o n t e n t of t h e pancreas i n c r e a s e d and remained e l e v a t e d up t o 96 h o u r s a f t e r CUB

I4

treatment. GSH is involved in the detoxication of various xenobiotics, conjugating foreign compounds to make them available for excretion in the bile and urine. The authors reported that ultrastructural and histological changes seen iq the pancreata of CHB treated rats were indicative of apoptosis and not necrosis. The GSH content of the liver was also determined after CHB dosing, and there was a slight depletion in GSH at 1. 6, and 24 hours after dosing followed by elevations of GSH at 48 and 72 hours. No hepatic lesions were found in the livers of treated rats at the 200 mg/kg daaa used.

Ahmed end Perooqui (1982) determined that the toxicity of aliphatic nitriles is not dependent on cyanide release, but the degree of toxicity depends on the degree of unsaturation of the compound. Nitriles studied included acrylonitrile, acetonitrile, and fumaronitrile.

silver et a1 (1982) examined the metabolism of nitriles in vivo to cyanide. They determined showed intreperitonesl inj~ctions of unsaturated nitrilss resulted in less thiooyanate excretion as compared to that observed after oral administration of the same nitriles. Overall, the thiocyanate excretion associated with dosing of unsaturated nitriles was lower than that of saturated nitriler. The authors indicated that their results suggested that a number of factors are important in the release of cyanide from nitriles including the position of double bonds, route of

15

administration, length of the oarbon chain, and presence of substitutions at the a-carbon.

The mechanism of pancreatotoxic action of CUB is not known. An discussed above, cyanide release from CHB is not thought to be a major factor in its toxicity.

1.2. Metabolism of Xsnobiotics

Xenobiotics are compounds that are foreign to biological ryrtemr and are often harmful to living cells.

Xenobiotios include compounds found in nature, pollutants, and other chenicals such as drugs. The majority of these compounds ere lipophilic. Exposure to renobiotics can occur through dermal absorption, inhalation, or ingestion.

To cope with harmful xenobiotic exposure, organisms have developed a number of metabolic pathways which act on xenobiotics so they may be excreted by regular routes. he organisms are exposed to a variety of chemicals, they possess a number of different enzymes which are involved in the metabolism of xenobiotice (Armstrong, ,987).

Lipophilic compounds pose the greatest threat t o organisms as they can become widely distributed throughout the organism in lipid stores and membranes. Furthermore, they tend to remain in these lipid stores for long periods of tine. To ba excreted from the organism, they must therefore be transformed to more hydrophilic molecules which can be readily eliminated (Armstrong, 1987).

16

Biotransformation reactions taka place mainly in the liver (Chhabra and Eastin, 1984). but other organs such as the brain, kidneys, adrenaln, small intestine, and the pancreas have xenobiotic mstsbolirm oapability.

Biotransformation can occasionally result in the inadvertant retention of xenobiotics by causing the formation o r more reactive metabolites which eaact with endogenous macromolecules (Caldwell, 1985).

There are two classes of biotransformation reactions, known as phase I and phase I1 reactions. Phase I reactions modify the molecular structure of the xenobiotics by oxidation and reduction reactions. These reactions are carried out by a number of related hemoproteins, notably the c y t ~ ~ h r ~ n e P-450 mono-oxygenare system. Other oxidative reactions are carried out by aldehyde and alcohol dshydrogenaser (Laitinen and WalKins, 1985).

Phase I1 reactions involve the addition of endogenous Substrates which are hydrophilic and increase the solubility of the compound, enabling it to be excreted in the bile or urine.

There are seven major phase 11 reactions of xanobiotica occuring in namalr. These are glucuronic acid, sulfuric acid, glutathione, and amino acid conjugations, and also methylation, aoetylation, and eporide hydration reactions (Caldwell, 1985; Sipes and Gandolfi, 1986).

only GSH conjugation will be d i b ~ ~ s s e d here as it has

17

been implicated in the detoxioation of unsaturated aliphatic nitriles.

1.2.1. alutathione and Glutathione 8-~ranaierase GSH is a tripeptide composed of L-qlutamate, L- cystsine, and glycine (Figure 1-31, GSH exists in 2 forms, as a thiol reduced molacule (GSH) and in a dirulfide oxidized (GSSG) Corn. The ratio of GSH:GSSG in the liver is maintained at 250:l. It is found in nearly all tissues of aerobic organisms and is usually found in rnillimolar concentrations (Kaplowitz et al, 1985). GSH is present mainly in the cytosol of cells and is the nost abundant sulfhydryl in animal tissues (Chasreaud, 1979).

One of the highest concentrations of GSH in the rat is found in the liver (approximately 172 mq/100g tissue). A substantial amount OK GSH is also found in the pancreas (approximately 56 mg/lOog tissue) (Boyland and Chaeseaud, 1969).

The GSH molecule is of biological importance because it is a specific substrate for GSH transfereses (GsH-T), a group of enzymes that play an important role in the detoxioation of foreign compounds (Kaplowitz et al, 1985).

GSH transferases (a family of isoenzynes) conjugate nucleophilic GSH to lipophilic conpounds that have an electraphilic center. Many of these conjugations occur nonenzymatically, but the action of GSH transferaaes

7Glu I Cys - ^SH

I

GlY

2 0

inoreases the rate of the reaction (Chassaeud, 1979).

GSH transrerases bind the lipophilic compounds and GSH by lowering the pK. (9.3) of GSH which results in the ionization of GSH to 0% and H'. Tha 0% ion is more nuslaophilic than GSH and is mare likely to bind to the electrophilic center of the bound substrate molecule (Keen et a l , 1976). The resulting compound is a thiol ether.

Different GSH transferares are responsible for the conjugation of GSH with different classes of lipophilic compounds. The isazper are specific with respact to organic groups in the electrophilic substrates such as alkyl, aryl, and epoxy groups (Mannervik st al, 1983). GSH tranaferaeer can oonjugata suitable substrates that are both endogenous and exogenous, but a major function is their role in detoxifying reactive molecules that nay react with biologioal nalecules such as proteins and DNA.

In addition to their enzymic activity, GSH transferases are important in funotioning as intracellular ligands. Far example, ligandin or Y protein of the hepataoyte is important in binding and intracellular transport of bilirubin (Ketterer et al, 1988).

Xenobiotics may be reactive as the intact forms, or they may be activated following phase I biotranrfornation.

GSH transferahes are one of a number of detoxifying phase I1 enzyme systems responsible for detoxifying such molecules.

Some reactive molecules are known to initiate carcinqenesis

21

and nutaganesis. Therefore the ability of GSH transferases t o detoxify remotive elactrophiler is of vital importance, as is the case concerning the detovicatian of acetaminophen where small amounts of a reaotive quinone inine are potentially very injurious (Ketterer, 1988).

Other drtoxifying reactions of GSH transferases involve their ability to act ass GSH peroxidasor and detoxify hydroperoxides. Hydroperoxides are harmful to tissues

(Nosialou and Morgenstern, 1989). forming GSSG which is later reduced hy GSH reductase. Hydroperoxides which are f o m a d from lipids during oxidative stress can propagate free radical formation within cells, and have been implicated in cercinogenesis (Ketterer, 1988).

1.2.2. Heroapturio Aoid P m m a t i o n

After glutathione oonjugation occuring in the liver, the metabolites are transported into plasma and hila (Andere et al, 1988). The enryma -glutamyltransferase (EC 2.3.2.2.) catalyses the removal of the glutamyl portion of the conjugate. Glycine is then removed from the molecule by cysteinyl glycine dipeptidaees (EC 3.4.13.6. and EC 3.4.11.2.1, leaving a cysteine S-conjugate (a thial ether).

Both processing of plasma GSH and cysteine S-conjugates is carried out primarily in the kidney, which metsbolises GSH conjugates as described above. cysteine S-conjugates are acetylated by N-aoetyltransferaee (EC 2.3.1.80.) to form

22

mercapturic acids which are water-soluble and evoreted in the urine (Wood, 1970; Anderr et a l , 1988) (Figure 1-4).

1.1. Pancroatotoxina

The pancreas, although primarily an endocrine and exocrine functioning organ, is capable of a number of biotransformation reactions (Scarpelli, 1989). These reactions can both protect it from xenobiotio denage or cause harm by creating more reactive metabolites, as also seen in the liver and other organs.

Toxins that have selective toxicity for the panoreas are not o o m n , and hence CHB is a very interesting compound because of its selectve panoreatotoricity. there is no commonality among pancreatotoxins. Pancreatotoxins cause acinar cell necrosis and other injuries, depending on the intensity, duration, a-d frequency of exposure.

Panareatotoxins include alcohols (such as ethyl and ally1 alcohol), amino acids and their derivatives (i.e.

azaserine), aulEonamides, and estrogens (Scarpalli. 1989).

carcinoma of the exocrine pancreas was fifth among cancers as a cause or death in the United States in 1983 (Wiebkin et el, 1984). The pathogenesis of this disease is not known, but several pancreatic carcinogens are currently being investigated. compounds known to induce pancreatic censer include the nitrosamines N-nitrosobis(2- ompropy1)anine (Kokkinakis, 1983) and N-Nitoso-2,s-

Bigura I-4% Me~eapturio M i d Formation

25

dinethylnorpholine (Soarpelli et el, 19821, which are carcinogenic t o the hamster panoreas. Aaaserine, an amino acid derivative, is a rat pancreatic carcinogen, and has been extensively studied by Longneoker and his colleagues

(Longneoker et a1, 1981). It is not known if cHB is carcinogenic.

24

l Glutathlone YGlu

I

Cys-SH

+

YGlu I Cys-S-R

I

1

I l l Glutathlone Conjugate

hydrolysis

acetylatlon

2 6 1.4. Objectives of the present study

m view of the evidence quoted above that the natural (s)- enantioaer of 1-cyano-2-hydroxy-3-butene is a pancreatic toxin in rats, the following objectives for the present study were developed.

1. To determine the effect of a synthetic 5 0 : 5 0 enantiomeric mixture of cns on pancreatic and hepatic non- protein thiol concentrations.

2. To determine the inducibility of pancreatic and hepatic glutathione s-tranaferase by synthetic M E .

3. TO determine the effect of synthetic CHB on pancreatic juioa secretion and on the protein concentration of pancreatic juice.

4. TO investigate the effect of synthetic CHB on bill secretion and bile eoid oonoentration.

5. TO examine the excretion of [lac] synthetic CHB in the rat and its possible aocunulation by the pancreas.

chapter z naterials and Methods

2 . X . Synthesis Of CBB

CHB was derived synthetically as a racemic mixture of ( R ) - and (S)-CHB as described by Das and Torsell (19841, with some slight modifioation. 1,3-~utadiene is the starting compound used in the synthesis, which is parformed in 2 steps (Figure 2-1). The first step produces 5-vinyl-2- iroxaaoline, which is then used to produce CHB. All chemicals were obtained from the Aldrich chemical Company I n c . (Ws,usA) ald were of the highest purity available.

2.1.1. synthssia oz 5-vinyl-2-isouaaoline

Approximately 5.2 g ef 1.3-butadiene was dissolved in 48 mL of benzene:acetonitrile (2:l). At O'C 4.4 q of nitromethane, 7.0 g of triethylamine. end 7.8 g of chlorotrimethylsilane were added to the 1.3-butadiene in benzene:acetonitrile. The mixture was stirred for 3 hours at 0-C, then at room temparature for 1 days, at which paint

30

0.5 mL of teifluaroacetic acid was added. The mixture was stirred at room temperature for another 24 hours. he final mixture wan an orange-brawn ealution containing a light brown precipitate. The precipitate was filtered and washed with lo nL of benzene and then with 30 mL of water. The filtrate was then dried with anhydrous magnesium sulphate, filtered, and excess solvents were evaporated from the remaining filtrate. The filtrate war 5-vinyl-2-isouazoline, a dark orange-brawn liquid.

2 . 1 . 2 . Spthenis oC CBB from 5-vinyl-2-isoxa~oline 5-vinyl-2-inouazoline (1.8 g) was refluxed with 4.0 g of tristhylamins in an oil bath at 100°C for one hour. The exoesc triethylamine was evaporatad and the mirtura distilled in vacuo. At e pressure of 6 mmHg CHB had a boiling point of approximately 63OC and distilled as a pala yellow liquid. Approximately 1-1.5 g of CHB was obtained from eaoh synthesis. StrUotUre of cns was confirmed by ['HI NMR (CDC1,I. The spectrun showed that the conpound was essentially pure, contaminated slightly by acetonitrile.

3 1

2.2. Uon-Protein T n i o l and G l u t a t h i o n e S-~ransferaae Induetion

2.2.1. animals

Male Sprague-Daulsy r a t s weigh:ng 235 t o 280 g were purchased from Canadian Hybrid Farms INS, Canada). The r a t s were housed i n p l a s t i c r e c t a n g u l a r Nalgena =ages with removable l i d s l i n e d w i t h sawdust (Beta-Chip, C h a r l e s R i v e r , st. C o n t a n t , PQ) (3 o r 4 r a t r l o a g e )

.

and 40% r e l a t i v e humidity. The animals were provided w i t h food ( P u r i n a R a t Chow 115012) end t a p w a t e r ad libitum u n t i l t h e day o f s a c r i f i c a .

2 . 2 . 2 . Treatment w i t h cHB

s y n t h e t i c M B was e m u l s i f i e d i , o l i v e o i l to a P i n a l c o n c e n t r a t i o n o f l o o mg CHB/mL a l i v e o i l . R a t s r e c e i v e d CHB i n o i l i n t r a g a s t r i c a l l y u s i n g a 5Prench i n f a n t n a s o - g a s t r i c f e e d i n g t u b e (Medi-Craft Limited, ON, Canada). The dose of CUB i n o i l r e o e i v e d was 200 mg/kg body w e i g h t ( 2 . 1 mmol/kg) which was approximately 0.5 nL. Control animals were dosed w i t h o l i v e o i l a l o n e ( 0 . 5 mL). The animals were l i g h t l y a n a s s t h a t i z e d j u s t p r i o r t o d o s i n g with d i e t h y l e t h e r .

Animals used i n t h e t i m e course experiment were s a ~ r i f i ~ e d a t 0 hour and a t 4, 24, and 120 hours a f t e r

3

3 1 I

dosing. Six animals warm used for each treatment group and the 0 hour group war used as a control.

2.2.3. Cell Praetionation and Protein Preaipitation

m t s were sacrificed by exranguination from the j abdominal aorta under diethyl ether anaesthesia. The

abdominal cavity was flooded with chilled 0.9% NaCl (saline) solution. Approximately 500 ag of liver and the entire pancreas were removed and placed in ice-cold saline. The pancreas; was trimmed of remaining fat, major blood vessels.

and connective tissue. Both the liver and pancreas were blotted between two layers of Na.2 filter paper (Whatman) under a 200 g weight for 15 secandr to remove surface saline. Approximately 200 mg of hepatic tissue (from the bottom right lobe of the liver) and 400 mg of pancreatic tissue from each animal Were taken and placed into beakers containing 25 mL of oold Tris buffer (50 mM Tris vith 0.25 M sucrose, pH 7.5) for GSH-T and protein assays. For homogenization, samples were poured into 30 mL glass homogenization tubes (wheaton, USA).

Por determination of "an-protein thiol (NP-SH)

approximately 100 mg of hepatic tissue and 200 ag of I pancreatic tissue iron each animal war taken and placed in a test tube containing 5.0 m~ of 0.02 M EDTA. For homogenization, samples were poured into 10 mL glass

33 homogenization tubas (Wheaton, U S A ) .

All tissues were homogenieed at 0-4-C using a teflon pestle attached to an electric 3/8" drill (Black and Decker). The pestle was moved up and down nine tines for homogenization of pancreatic tissue, and six timer for homogenization of liver tissue at e pestle speed of 1200 rpm. The vertical movement and rotation of the pestle results in a tearing and mincing of the tissue and agitation of the cell= generating a horogenate conposed of dilutad cell cytoplasm, intraoellular particles, end some unbroken Cells.

The GSH-Tlprotein homoganater were then transferred to Beckman polyoarbonate oentrifuge bottles l"X3.5" and centrifuged for 60 minutes at 105.000 g,.,, in a ~ e c k m a n LS-65 type H ultracentrifuge (35,000 rpn, 50.2 Ti rotor, I'c).

TWO 3 mL samples were placed in 7 rnL glass scintillation vials, one vial placed in ice for protein assays, the other stored at -7O'C until assayed for GSH-T activity. nfter samples were removed far protein assays, the first vial was also stored at -70DC. Duration of storage never exceeded 7 days.

The NP-SH homogenates were usad immediately for the NP- SH assay.

3 4 2.2.1. Peinoiple of Protein Assay

~sti~nation of sample protein concentration was executed using the ~io- ad protein assay kit, a dye-binding assay (Bio-Rad, ON). The assay is based on the differential colour change of a dye, in this case Coomassie Blue G-250, in response to various protein oonoentrations. The absorbance maximum of this dye shifts from 465 nm to 595 nm when binding to protein occurs (Pierce and suelter, 1977).

and this inorease is monitored spectrophotonetrically.

2.2.4.1. Preparation of Dye Reagent

The dye reagent was provided as e five-fold concentrate whioh was diluted with deionized watar and filtered through a N0.1 Whstnan filter paper into a glass beaker just prior to use. The reagent was newly prepared every time the assay was performed and kept at ruom temperature until used.

2.2.1.2. Protein standard

The protein standard used was Bio-rad Standard I, lyophilized bovine g a m a globulin which was reconstituted with 20.0 mL of deionized water. The final concentration of this standard war 1.4 mg/mL. It was stored at 4 - C for up to 60 days

35 2.2.1.3. Pl~teill Assay Method

Volumes of 10, 20, 40, 60, 80, and 100 pL of the Standard protein eolution were added to 4.5 mL polystyrene d i s p o b a b l ~ CUVBttes. This yielded final protein concentrations of 5.82, 11.6, 23.0, 31.2, 45.3, and 56.2 pg proteinlml after 2.5 mL of diluted Bio-rad reagent was added using an Eppendorf repeater pipetter. The standards and unknown samples were mixed by gentle inversion. Samples were allowed a 5 minute equilibration pariod and then were read at 595 nm in a ~ i l f o r d 250 speccrophotometer against a blank containing only diluted reagent. The absorbance readings of the known c o n ~ e n t r a t i o n s were used to oonstruct a standard curve (protein concentration versus absorbance).

T h e curve was then usad to determine protein concentration of the unknown samples. A new standard curve was constructed each time the assay was performed, and all assays were done in triplicate.

2.2.5. Prinoiple of Uon-Protein Tlliol Lssay

GSH is the major free thiol in most living cells and thus GSH levels were determined using the non-protein sulfhydryl assay bared on the method of Ellnen (1958) and modified by Sedlak and Lindsay (1968) (Figure 2-21, Ellman reported that 5~.5r-dithiohis-(2-nitr~hbb~~ii acid) (OTNB) is reduced by 1 mole of aulfhydryl group to farm 1 mole of

pig".. 2-2: The reacticm of 5Ur5'-dithiobis-(2-nit~0bb~~0i0 ..id) ( o w e ) with a evlfhydryl group to for*

2-nitr~-~-mer..r.llptobenzoiC acid

S

1 \

I qoH

^\

R-SH

+ 4

4

. S Q N o 2

-

COOH COOH

38

2-nitr0-5-mercapt0benzoic a d d . The resulting nitromarcaptobenzoic acid anion has an intense yellow colour, and its absorbance ia measured

spectrophotometrically. By creating a standard curve of the conoentration of CSH versus absorbance, determination of NP- SH concentrations in unknown samples is possible.

2.2.5.1. Standard Preparation

A standard N r v e of GSH concentration versus absorbance was oonstructad using concantrations of 1 to 10 ( x l O J ) M of GSH (sigma, USA). The GSH was dissolved in deionized water.

The GSH was added to 0.4 M ~ r i s buffer at pH 8.9 in a test tube to yield a final volume of 6.0 mL, then 0.1 mL of 0.01 M DTNB in absolute mathano1 was added. The tube was inverted to mix the contents, then the contents were tranferred to a cuvatte and the absorbance read at 412 na within 5 minutes of the DTNB addition. Buffer with DTNB added served as a blank.

2.2.5.2. Determination of UP-SA Qroups in samples After homogenization in 0.02 M E ~ A , the 5 la^

homogsnatas were placed in 15 mL test tubes, and 5 aL of 10%

TCA was added t o each tube to precipitate the protein present. The tubes were allowed to stand for 15 minutes at room temperature with intermittent shaking, then centrifuged

39

f o r 20 minutes a t 3000 g i n a Clay Adams (NJ, USA) Dyanc I1 C e n t r i f u g e . Two rL o f t h e s u p e r n a t a n t was added t o a t e s t t u b e o o n t a i n i n g 4.0 mL of 0.4 M T r i s b u f f e r a t pH 8.9. hen 0 . 1 mL of a 0 . 0 1 W s o l u t i o n of UTNB i n a b s o l u t e methanol was added, t h e c o n t e n t s mixed and t r a n s f e r r e d t o a polycarbonate c u v e t t e . The absorbance. of t h e c u v e t t e s were read a t 412 nm w i t h i n 5 m i n u t e s of t h e a d d i t i o n of DTNB a g a i n s t a r e a g e n t b l a n k c o n t a i n i n g no honogenate.

2 . 2 . 6 . P r i n o i p l e of ~ l u t a t h i o n e - ~ - ~ r a n s f e r 1 1 9 e ~ c t i v i t y xssay The a s s a y t e c h n i q u e u s e d was t h e method r e p o r t e d Ily Xabig rt ^{a1 (19741} a s modified by Kraus and K l o f t (1980).

The a s s a y d e t e r m i n e r GSH-T a c t i v i t y by measuring t h e change i n absorbance a s a GSH-substrate c o n j u g a t e is formed by t h e enzyme.

The s u b s t r a t e u s e d i n t h i s assay is l-chloro-2.4,- d i n i t r o b e n z e n e (CDNB) and is t h e most common s u b s t r a t e used f o r t h i s a s s a y . CDNB i n e t h a n o l h a s a s l i g h t yellow c o l o u r , b u t when c o n j u g a t e d t o GSH, t h e p r o d u c t i s a l i g h t - i l k y w h i t e s o l u t i o n ( F i g u r e 2-3). The g r e a t e r t h e e o t i v i t y of GSH-T p r e s e n t i n t h e t e s t sample, t h e g r e a t a r t h e chango i n absorbance w i l l be d u r i n g t h e a s s a y .

2.2.6.1. GSH-V A e t i v i t y assay Bsthod

The b u f f e r used i n t h e assay was 1 0 mM phosphate b u f f e r

Piguee 2-31 The rsaotion or 1-chloro-2,l-dinitzobennnne ICDNB) with GSR to t o n a glutathione eonjugate

O~NQCI

GSH - ^O~N-@G

^Hcl

NO2 NO2

4 2

( p ~ 7 . 4 ) c o n t a i n i n g 1.1 mM magnesium c h l o r i d e , k e p t warm a t 3'1-C. he volume o f l i v e r c y t o s o l assayed was 2 0 In and t h e volume of p a n c r e a t i c c y t o n o l assayed was 100 pL. A G i l f o r d 250 s p e o t r o p h o t o m a t e r c o n t a i n i n g a 4 chambered w a t e r - j a c k e t e d c u v e t t e h o l d e r was used f o r t h i s a s s a y , e q u i l i b r a t e d a t 3 7 T .

Phosphate b u f f e r was p i p e t t e d i n t o t h e c u v e t t e a p l a c e d i n t h e spectrophotometer, then t h e a p p r o p r i a t e volume of sample was added t o t h e c u v e t t e s , w i t h t h e f o u r t h a c t i n g as a blank, c o n t a i n i n g no sample. B u f f e r war added t o y i e l d a f i n a l volume o f 3 mL i n a l l c u v e t t e s t a k i n g i n t o a c c o u n t a d d i t i o n s of GSH and CDNB l a t e r . 100 uL of GSH i n w a t e r was added t o a l l c u v e t t e s ( a a d o up t o y i e l d a f i n a l

c o n c e n t r a t i o n of 1 mM i n 3 mL). The samples were i n c u b a t e d a t 37*C i n t h e spectrophotometer f o r 5 minutes, t h e n 50 pL of CDNB i n e t h a n o l was added (made up t o y i e l d a 1

m

s o l u t i o n i n 3 mL) t o each m v e t t e and t h e c o n t e n t s of e a c h o u v e t t e mixed.

The absorbance change war measured a t 3 4 0 nm and r e c o r d e d on a G i l f o r d 6050 c h a r t recorder f o r 3 lninutes a f t e r t h e CDNB a d d i t i o n . The a v e r a g e of t h e t h r e e enzyme a o t i v i t i e a was o a l c u l a t e d using t h e e x t i n c t i o n c o e f f i c i e n t f o r CDNB as 9.6 m ~ ' o m ' and expressed as naol GSH conjugated/min/ng p r o t e i n .

4 3

2.3. *he effect o f CUB on Panoreatio m i c e and siis secretion

2.3.1. animals

Male Spraque-Dawley rats weighing 2 3 5 to 325 q were obtained from Canadian Hybrid Farnr (NS, Canada) and maintained as previously described.

2.3.2.1. Anaesthesia

Diathyl ether vapour was used to induce and maintain anaesthesia. The rat was placed in a glass jar containing cotton soaked with ether, and the jar covered with a heavy plsriqlas lid. After anaesthesia had been achieved the rat was removed. A 150 mL beaker aontaining gauze soaked with ether was used as a mask t o maintain anaesthesia during the surgery.

2.3.2.2. Bile mct cannulation

The rat was placed in the supine position and the abdomen shaved with an electric shaver. The abdomen was opened by a midline laporotony incision 3.5 cm long using a No.10 blade.

The duodenum was deflected t o expose the common bile

4 4

duet and the duodenal head of the pancreas. The cornon bile duct was cannulatad at the ampullae of Vater with polyethylene tubing (PE 10, Clay Adens. 0.0.=0.61 mm, 1.0.=0.28 mm) measuring 18 cm long. The cannula was inserted into a small incision in the duct and secured with 2 silk sutures (Ethiccn, 4-0 silk). This cannula collects a mixture of bile and pancreatic juice but after cannulation of the common bile duct above the Fanoreas it collects pure pancreatic juioe (Figure 2-4).

The common bile duct above the pancreas was also cannulatea by inserting another 18 om long PE 1 0 tube into the duct. This cannula war used t o oollect bile.

Both cannulae ware exteriorized through stab wounds in the aide of the abdomen.

2 . 3 . 1 . 3 . Duodenal Cannulation

A 5French infant naro-gastric tube, 45 on long, was placed in the duodenum approximately 8-10 nm distal to the pylorus (Figure 2-5). The cannula was inserted into a m a l l incision and secured with a oyanoacrylate tissue adhesive.

The tubing was exteriorized through a stab wound in the right flank of the abdomen. T o ensure that administered doses are quantitatively delivered t o the duodenal lumen and as a check against leakage, approximately 0.5 m L of 0.9):

NaC1 was infused through the cannula.

~ i g u r e 2-41 B i l e m o t Cannulation

Pigure 2-5: Duodenal cannulation

49 2.3.2.4. Closure

T h e abdominal organs were returned to their proper p o ~ i t i o n a and the muscle layers and skin closed seperatsly With a continuous silk suture (Ethicon. 4-0 ~ i l X ) .

2.3.2.5. Post-operative housing and sacrifice T h e animals were secured in Bollman restraint cages immediately after surgery as t h i s provides humane immobilization t o prevent access t o the cannulae. A saline- glucose solution (0.9% NaCl containing 5% glucosa) was infused into the duodenal cannula at a rate of 2.5 allhr during recovery (approximately 24 hours) using a Harvard pump. Food and water were witheld and the animals kept at a temperature of 23'C with a 12 hour photaperiad.

After the experiment, animals were anaesthetized by diethyl ether and sacrificed by exsanguination from the abdominal aorta. Animals that did not have constant pancreatic juice and bile secretion on the first post- operative day were eliminated fran the study.

2.3.3. Test Dosages and Administration

CHB was emulsified in 1 mL volumes of olive oil at the following doses: 25. 50. 100. 150, and 200 mg/kg body weight. Each test group for each dose contained six rats.

Experiments ware performed on t h e first port-operative day (

50

approximately 20 hours after surgery). At zero time, the saline-glucose infusion was stopped and the appropriate dose was administered as a pulse through the duodanal cannula (volume approximately 0.5 mL). Approximately 0.5 mL of saline ( o . 9 t NaC1) was then passed through the oannula ensuring that the test dose had been delivered into the duodenum. The intraduodenal glucose-saline infusion was then restarted at a rate of 3 mL/hr with the infusion pump.

2.3.4. Pancreatia Juice and Bile sample Colleotions Pancreatic juice and bile were oollected in preweighed plastic tubes end glass vials, respectively. Both Were collected at half hour intervals for sevan hours. Control samples of pancreatic juice and bile ware collected at one hour and 3 C minutes before administration of the test dose.

Tubes and vials were re-weighed to determine the mass of pancreatic juice and bile oollected. Both pancreatic juice and bile were assumed to have a density of I g/nL.

2.3.5. Determination of Protein Conoentration in Panareati.

Juice

Protein concentration in collected panareatic juice was determined using the Bio-rad assay previously desoribed. As pancreatic juice has a very high protein ooncentration, each sample was diluted five tines for the assay with deionized

51 water.

2.3.6. Principle of Bile Acid Concentration ~ s s a y The majoe bile acids are taurine and glycine conjugates of cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic a d d s . 1n ths determination of bile acid concentrations, the bile acids are first oxidized to )-or0 bile acids by 3.-hydroxyateroid dehydrogenase (3o-HSD) along with the reduction of NAD to NADH. NADH is then oxidized t o NAD with tha reduction of nitro blue tetrezaliue (NET) to formazan by the ennyne diaphorase (Nashige st al, 1981).

Formazan has a maximum absorbance at 530 nm and the intensity of the colour produced is directly proportional t o the concentration of bile acids present.

2.3.6.1. Preparation of Reagents

Reagents were supplied in the Bile Acids Kit (Sigma, USA). Two types of reagents were used in this assay.

Reagent A contained NAD (2.5 mmcl/L), NET (0.61 mmol/~), diaphorase (625 unita/L). and buffer (pH 7.0). Reagent B contained 30-HSD (1250 unitell).

Reagents A and B were reconstituted with the app~-opriate amount of water. Test reagent was prepared by combining 4 m L of Reagent A with 1 m L of Reagent 8 . Blank reagent was prepared by combining 4 mL of Reagent A with 1