Materials and methods

M elfo rd L a b o ra to rie s L td ., Ip s w ic h , UK

(3-nicotinamide adenine dinucleotidePhoshate (NADP) and (3-nicotinamide adenine dinucleotide phoshate reduced (NADPH)

Sigm a - A ld rich , D o rs e t, UK

Ammonium persulphate, secondary a n ti-rab b it monoclonal antibody, secondary anti­

goat monoclonal antibody, bovine serum album in, l-c h lo ro -2 ,4 -d in itro -b e n z e n e (CDNB), 3, 4-dichloronitro-benzene (D C N B ), 5, 5' - d ith io -b is (2 -n itro )- benzoic acid (D TN B ), 7 - ethoxycoum arin, ethoxyresorufin, D-glucose, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, glutathione reductase, glutathione, 7-hydroxycoum arin, glycerol, (3- glucuronidase, hydrocortisone-21-hem isuccinate, HRP-conjugated an ti-ra b b it and a n ti­

goat secondary antibodies, insulin, p-m ercaptoethanol, 7-m eth o xyresorufin , 2 -m e th y l-1,4-naphthoquinone (m en ad io n e), 3-(N -m orpholino)propanesulforic acid (M O PS), tween 20, thiazoyl blue tétrazolium bromide (M TT), 7-pentoxyresorufin, polaroid film , pyronin Y, resorufin, D-saccharic acid, sodium dodecyl sulphate (S D S ), sulphatase and N,N,N ' ,N ' -tetram eth yleth ylen e dia m in e (TEMED)

O xoid Ltd, B as in g s to k e , UK Oxoid nutrient broth No. 2

Roche D iag n o stics, M a n n h e im , G erm a n y Cytotoxicity detection kit (LDH)

RSCH P h arm a c y, G u ild fo rd , UK Sterile IV sodium chloride 0 .9 % (5 0 0 m l)

Chapter 2: Materials and m ethods

2 .2 M eth o d s

2 .2 .1 A n im a ls

Male W istar albino rats (2 0 0 -2 5 0 g ) were purchased from Bantin and Kingman (B&K) Universal Ltd (Hull, UK). The animals w ere housed on sterilised sawdust in solid bottomed polypropylene cages, with 2 -4 rats per cage except experim ents involving urine collection w here anim als w ere housed singly in m etabolic cages for no longer than 4 days. The am bient tem p eratu re was m aintained a t 2 2 °C ± 3 °C and hum idity controlled in the range of 3 0 -7 0 % . Lighting was artificial, with a 12h lig h t/12h dark cycle. W a te r and standard dry rodent food in the form of pellets was freely available at all tim es. The anim als w ere allowed to acclimatise to th e above conditions for a t least 24h before sacrificing.

2 .2 .2 P re p a ra tio n a n d m a in te n a n c e o f p re c is io n -c u t tis su e s lices

Precision-cut tissue slices, of uniform thickness and d iam eter, w ere cut from hum an liver and rat liver and lung tissues and incubated under controlled conditions as described by Hashemi et al. (1 9 9 9 a ).

2 .2 .2 .1 P re p a ra tio n o f liv e r slices

Im m ed iately a fte r sacrifice of rats by cervical dislocation, livers w ere excised and placed in ice-cold EBSS enriched with D-glucose (2 5 m M ) and gassed with 9 5 % o x yg en / 5 % CO2 for Ih . The liver lobes w ere cut free. With respect to the hum an liver, the tissue,

delivered as 1 7 0 -2 0 0 g single piece, was cut into 1 cm thick sections using a scalpel.

Tissue cylinders w ere cut with a hand-held coring tool (V itro n , Tuscan, US) to a d iam eter of 8 mm from the sections of human liver or whole lobes of rat livers. The tissue was kept subm erged in ice-cold EBSS throughout the procedure. Liver slices (8 m m in

Chapter 2: Materials and methods

Krumdieck tissue slicer (Alabam a Research and D evelopm ent corporation, Alabam a, US) filled with chilled (4 °C ) oxygenated EBSS. The parts of the slicer th a t cam e into contact with the buffer and the tissue w ere wiped with 7 0 % ethanol prior to use. The slicer was set to operate in the in term itten t mode with a cycle speed of 4 0 cycles/m in in order to minimise mechanical trau m a to the tissue.

2 .2 .2 .2 P re p a ra tio n o f lung slices

The process of preparation of lung slices is sim ilar to th a t of the liver, but with a few differences. The first difference was th a t the lungs had to be stabilised with agarose. For this purpose, lungs w ere slowly infused with 0 .7 5 % solution of w arm (3 7 ° C) low- melting point agarose (4 8 m l/k g body w eight) through a cannulated trachea (IV cannulae Venflon 2 0 G ). The trachea was clamped and the lungs w ere kept subm erged in ice-cold (4 °C ) EBSS until the agarose solidified. At this point, the lobes of the lung w ere cut free and tissue cylinders w ere prepared in the sam e w ay as discribed for the liver. The second difference was th a t the thickness of the lung slices, set a t 4 0 0 -6 0 0 pm , was double th a t of the liver. The thickness of the lung slices, optim al for th e ir viability during the m aintenance in the culture m edium , was determ ined experim entally (U m achandran and loannides, 2 0 0 6 ).

2 .2 .2 .3 P re p a ra tio n o f c u ltu re m ed iu m

Tissue slices w ere p re-incubated in the culture medium prepared as described by Lake et al. (1 9 9 3 ), and consisted of RPMI 1640 supplem ented with insulin ( Ip M ) , L- m ethionine (0 .5 m M ), hydrocortisone-21-Hem isuccinate (O .lm M ), foetal bovine serum (5 % v /v ) and gentam ycin (5 0 p g /m L ). The incubation m edia contained, in addition, a series of concentrations of the test com pound(s) prepared in DMSO. The final concentration of DMSO was equal in all tre a tm e n t groups and did not exceed 0 .5 % (v /v ).

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Chapter 2: Materials and m ethods

2 .2 .2 .4 In c u b a tio n o f tis s u e slices

The tissue slices w ere transferred into 12-w ell culture plates (D o g tero m , 1 9 9 3 ), with each slice occupying a separate well containing the described above culture m edium (1 .5 m l), and w ere m aintained at 3 7 °C in an incubator equipped with an orbital shaker (S tu art orbital shaker, Barloword Scientific Ltd, Staffordshire, UK), in a humidified atm osphere containing 5 % CO2 (G alaxy B CO2 incubator. Scientific Laboratory supplies Ltd, N ottingham , UK).

Tissue slices w ere initially pre-incubated under these conditions in order to allow the the damaged cells to slough from the viable tissue. The pre-incubation period fo r liver slices was 0 .5 h while the pre-incubation of the lung slices lasted 1 h in order to allow the agarose to m elt. Tissue slices w ere then transferred to a fresh culture m edium containing eith er test com pound(s) or relevant solvent and w ere incubated fu rth er for up to 24 h. At the end of the incubation period, the slices w ere rem oved from the culture m edium , briefly rinsed in KCL (0 .1 5 4 M ) containing Tris (5 0 m M , pH 7 .4 ) and homogenised in the KCL-Tris buffer (1 2 slices in 1.2 m l).

2 .2 .3 LD H le a k a g e

Lactate dehydrogenase (LDH) is an intracellular enzym e. LDH release from tissue slices into the incubation m edium was used as an indicator of cytotoxicity. The LDH

concentration was m easured using a cytotoxicity detection kit (Roche Diagnostics, Mannheim, G erm an y), according to the m anufacturer's instructions. Three slices w ere used per each concentration of the test com pound(s). On com pletion of incubation (2 4 h), the culture m edium was aspirated and the rem aining tissue slices w ere each homogenised in 1.5 ml of 0 .0 1 M phosphate buffered saline (PB S), pH 7 .4 . The media and hom ogenates w ere centrifuged at 2 0 0 0 x g for 5 min a t 4 ° C using a bench

Chapter 2: Materials and m ethods

supernatants w ere m ixed with 0 .1 ml of freshly prepared reaction m ixture (1 1 .2 5 ml of dye w ere mixed with 0 .2 5 ml of catalyst) using a 96-w ell plate. The plate was incubated at room tem p eratu re for 15 min. The reaction was term in ated by addition of 0 .0 5 ml of stop solution and absorbance was read at 4 9 2 nm. Leaked LDH was expressed as

percent of total slice LDH released into the culture m edia (LDH m ed ium + LDH slice

hom ogenate = total LDH; LDH m ed ia/to tal LDH x 100 = % of total LDH released).

2 .2 .4 M e ta b o lis m o f 7 -e th o x y c o u m a rin

The 7-hydroxylation of 7-ethoxycoum arin by tissue slices was determ ined using the method of van lersel et al. (1 9 9 4 ) and Steensm a et al., (1 9 9 4 ). Liver or lung slices were incubated with 7-ethoxycoum arin (5 0 pM) for various tim e periods up to 6 hours. At the end of each tim e period, the incubation medium was aspirated and divided into three equal aliquots. The slices w ere rem oved, briefly rinsed in 0 .0 1 M PBS buffer, pH 7 .4 collected into 1 ml of NaOH (0 .5 M ) and sonicated (3 X 1 0 seconds bursts), allowing at least 1 m inute tim e intervals between the bursts in order to prevent overheating. The protein content of the sonicated slices was determ ined as described in section 2 .2 .6 .

Two of the aliquots of the aspirated incubation m edia, 100 pi in case of the liver and 4 0 0 pi in case of the lung slices, w ere incubated with Vi volum es of sodium acetate buffer (0 .5 M ), pH 5 .0 containing eith er (3-glucuronidase (5 0 0 0 U /m l) or a combination of sulphatase (2 5 0 U /m l) and D-saccharic acid 1,4-lactone (1 7 m M ), for 16h a t 3 7 °C in a shaking w a te r-b a th in a light-protected environm ent. The third aliquot was incubated under the sam e conditions but with Viz volum e of the sodium acetate buffer (0 .5 M ) only.

All three media aliquots and a series of known 7-hydroxycoum arin concentrations (0 -5 pM) w ere diluted to 1 ml with KCL (0 .1 5 4 M) containing Tris (5 0 m M ) buffer, pH 7 .4 ,

Chapter 2: Materials and methods

acidified with 2 5 0 pi of HCL (4 M ), and extracted with 6 ml of chloroform for 30 minutes.

At the end of the extraction process, an aliquot (5 m l) of the chloroform layer was transferred into clean test tubes and extracted with 3 ml of glycine-sodium hydroxide (0 .5 M ), pH 1 0 .5 for a fu rth er 30 minutes. The fluorescence of the aqueous layer was determ ined using an excitation wavelength of 3 80 nm and an emission wavelength of 4 52 nm. All samples and standards w ere analysed in triplicate replicates.

2 .2 .5 P re p a ra tio n o f s u b c e iiu ia r fra c tio n s

Hepatic and pulm onary subceiiuiar fractions w ere prepared as described by loannides and Parke (1 9 7 5 ). The tissue was kept at 4 °C throughout the procedure. On completion of the incubation, the slices w ere removed from the incubator, briefly rinsed in Tris (50m M ) buffer containing KCI (0 .1 5 4 M ), pH 7 .4 and homogenised (1 2 slices per replicate in 1.2 ml of buffer) in Tris (5 0 mM) buffer containing KCI (0 .1 5 4 M ), pH 7 .4 . The homogenates prepared from the tissue slices w ere centrifuged a t 9 0 0 0 x g for 20 m inutes a t 4 °C in eppendorf tubes using a bench m icro-centrifuge (Eppendorf, model 5 4 0 4 ). The whole livers, scissor-minced in three volum es of potassium chloride (1 .1 5 % w /v ) and lungs, scissor-minced in one volum e of potassium chloride (1 .1 5 % w /v ), w ere homogenised in the sam e w ay as the tissue slices and hom ogenates w ere centrifuged at 9 0 0 0 X g for 20 m inutes at 4 °C using a Beckman J2-21 floor centrifuge fitted with a JA- 17 fixed angle rotor (Beckm an Coulter Ltd, Bedfordshire, UK). The resulting supernatant (post-m itochondrial S9 fraction) was decanted, divided into aliquots and stored a t - 8 0 °C prior to analysis.

The S9 aliquots w ere thaw ed a t 4 °C , and microsomal and cytosolic fractions w ere prepared by centrifugation at 1 0 5 ,0 0 0 x g for 4 5 min a t 4 °C using a Beckman L8-70M floor ultracentrifuge and a fixed angle 70 IT I type rotor (Beckm an Coulter Ltd, Bedfordshire, UK). The supernatant (cytosolic fraction) was transferred to storage a t