All components of the assay except the substrate w ere pre-incubated for periods of tim e up to 1 h in a shaking w a te r bath at 3 7°C to facilitate the m etabolism of ITCs. The reaction was initiated by addition of an aliquot (1 0 pi) of ethoxyresorufin (0 .5 m M ) and the rate of resorufin form ation was monitored for up to 1 min a t 3 7 °C as described above.
2 .2 .7 .3 E ffect o f g lu ta th io n e on EROD a c tiv ity a fte r p re -in c u b a tio n w ith e ru c in /S F N
These studies w ere undertaken to determ ine w h eth er oxidized or reduced glutathione could reverse erucin/SFN-associated inhibition of rat C Y P IA I activity. EROD was assessed adapting the m ethod described in section 2 .2 .7 , the only difference being th at, once again, all com ponents of the assay except 7-ethoxyresorufin w ere pre-incubated in the presence of erucin/SFN (2 5 pM) and reduced or oxidized glutathione (5 -2 0 0 pM).
Each determ ination was carried out in triplicate. The following reagents w ere added in a fluorim etric cuvette:
Tris - HCL buffer (O .IM , pH 7 .8 ) 1 .90 ml
Microsomal suspension (2 5 % w /v ) * 0 .0 5 ml
NADPH (0 .5 m M ) 0 .0 1 ml
Test compound (1 .2 5 m M ) 0 .0 4 ml
GSH or GS-SG ( 0 .2 -8 m M ) 0 .0 5 ml
*m icrosom es w ere prepared from the livers of rats induced with Aroclor 1 254 as described in section 2 .2 .1 6 .4
All components of the assay except the substrate w ere pre-incubated for 0 .5 h in a shaking w ater bath a t 3 7 °C to facilitate the m etabolism of ITCs. The reaction was
Chapter 2: Materials and methods
initiated by addition of an aliquot (1 0 pi) of ethoxyresorufin ( 0 .5 m M ) and the rate of resorufin form ation was m onitored for up to 1 min at 3 7 °C as described above.
2 .2 .8 D e te rm in a tio n o f m ic ro s o m a l CYP3A a c tiv ity
7-Benzyloxyquinoline (7 -B Q ) is dem ethylated, predom inantly by CYP3A, to the fluorescent product 7-hydroxyquinoline (7 -H Q ). The assay was based on a method described by Stresser e t al. (2 0 0 2 ), and utilises 7-B Q as a m ark e r substrate for the CYP3A subfam ily of the cytochrom e P450 m ixed-function enzym e system . The following reagents w ere added to a fluorim etric cuvette:
M icrosom es W h o le liv e r /lu n g fro m tis su e slices m icro so m es
Tris buffer (O .IM , pH 7 .8 ) 1.75 ml 1 .9 5 ml
G lucose-6-phosphate (G -6 -P , 3 .3 m M) 0 .0 2 ml 0 .0 2 m l G -5-P dehydrogenase (0 .4 U /m l) 0 .0 4 ml 0 .0 4 ml
Microsomal suspension 0 .2 5 ml 0 .0 5 ml
7-BQ (4 m M ) 0 .0 1 ml 0 .0 1 ml
The reaction was initiated by addition of 0 .0 1 ml of NADP (1 .3 m M ) and the contents of the cuvette w ere m ixed by inversion. The rate of 7-H Q form ation was m onitored for up to 10 min at excitation and emission wavelengths set a t 4 1 0 and 5 3 8 nm respectively, the slit widths being set at 5 m m . The enzym e activities w ere calculated using a standard curve constructed by plotting fluorescence against 7 - HQ (0 to 0 .5 n m o le /m l).
2 .2 .9 D e te rm in a tio n o f N A D P H -C y to c h ro m e C re d u c ta s e a c tiv ity
This enzym e is localised in the microsomes and functions as a com ponent of the m ixed- function oxidase system . The activity is measured essentially by th e m ethod described by Williams and Kamin (1 9 6 2 ). The activity of NADPH-cytochrom e C reductase was
Chapter 2: Materials and m ethods
measured in the presence of either erucin or SFN using microsomes prepared from the livers of control rats. An aliquot (0 .1 m l) of each of the stock solutions of the relevant test compound (0 .0 3 , 0 .0 7 5 , 0 .1 5 , 0 .3 , 0 .7 5 and 1 .5 m M ), prepared in 5 0 % ethanol, was added to the reaction m ixture (3 m l) to achieve the required final concentration (1 , 2 .5 , 5, 10, 25 and 50 pM respectively). The solvent vehicle (5 0 % ethanol) was used as a control. The following reagents w ere added to both cuvettes:
T e s t c u v e tte R e fe ren c e c u v e tte Potassium phosphate buffer (5 0 m M ),
pH 7 .6 containing KCN (1 m M ) 1.8 ml 1.9 ml
Cytochrome C (0 .1 m M ) 1.0 ml 1.0 ml
Test compound (0 .0 3 - 1.5 m M ) or solvent 0 .1 ml 0.1 ml
Microsomal suspension 0.1 ml 0 .1 ml
An aliquot (0 .1 m l) of NADPH (3 0 m M ) was added to th e test cuvette to initiate the reaction. The contents of th e cuvettes w ere mixed by inversion. The increase in absorbance over tim e was followed at 5 50 nm for up to 2 min using a Kontron 8 60 spectrophotom eter. Enzym e activities were calculated using a m olar extinction coefficient of 1 8 .5 mM cm and w ere expressed in units of nmol per m inute per mg of microsomal protein.
2 .2 .1 0 D e te rm in a tio n o f to ta l g lu ta th io n e c o n te n t
The total cytosolic glutathione content was m easured essentially as described by Akerboom and Sies (1 9 8 1 ). An aliquot (0 .2 m l) of the cytosolic fraction prepared from whole livers or lungs (diluted 8 fold with KCI, 1 .1 5 % , w /v ) or tissue slices (undiluted) was mixed with perchloric acid (2 M; 0 .2 m l) containing EDTA (4 m M ) to precipitate the proteins. The m ixture was neutralised with potassium hydroxide (2 M; 0 .2 m l)
Chapter 2: Materials and m ethods
containing MOPS (0 .3 M ). Standards of glutathione w ere taken through the same procedure. All standards and samples, analysed in duplicate, w ere centrifuged a t 2 ,0 0 0 x g for 10 mins. The supernatant was decanted and used in the assay. The following reagents w ere added to a spectrophotom eter cuvette:
Potassium phosphate buffer (0 .1 M, pH 7 ) containing EDTA (2 .5 m M ) 1 .0 0 ml
NADPH (1 .5 m g /m l) 0 .0 5 ml
Glutathione reductase (6 U /m l) 0 .0 2 ml
Sample or standard 0 .1 0 ml
The reaction was initiated by addition of 0 .1 0 ml of 5 ,5 '-d ith io -b is (2 -n itro )-b e n zo ic acid (DTNB, 3 .8 m M ). The contents of the cuvette w ere mixed by inversion and the reaction was followed a t 4 1 2 nm for up to 2 min using a Kontron 9 3 2 spectrophotom eter. Total glutathione concentration was calculated from a standard curve constructed by plotting glutathione reductase activity against glutathione concentrations.
2 .2 .1 1 D e te rm in a tio n o f q u in o n e re d u c ta s e a c tiv ity
The assay was perform ed using the method described by Prohaska and Santam aria (1 9 8 8 ), which is based on measuring an increase in absorbance due to reduction of a yellow dye 3 -[4 ,5 -d im e th y lth ia z o ly l-2 -y l]-2 ,5 d ip h e n y l tétrazo liu m brom ide (M TT) to a
blue-coloured product, form azan, in the presence of the electron acceptor m enadione.
The following reagents w ere added in both spectrophotom eter cuvettes:
Tris buffer (2 5 m M, pH 7 .4 )
containing tw een 20 (0 .0 8 3 % , w /v ) MTT (1 0 m M )
T e s t c u v e tte
1 .0 0 ml 0 .1 0 ml
R e fe re n c e c u v e tte
1 .0 0 ml 0 .1 0 ml