A new method to study glutathione adduct formation in periportal and pericentral regions of the liver lobule by micro-reflectance spectrophotometry.

A method was developed to measure the formation of glutathione adducts of 1-chloro-2,4-dinitrobenzene (CDNB) and 2,4-dichloro-1-nitrobenzene (DCNB) in periportal and pericentral regions of the liver lobule in the isolated perfused rat liver by surface reflectance spectrophotometry. Conjugates of DCNB and CDNB are released from livers of normal and phenobarbital-treated rats during perfusion in either the anterograde or the retrograde direction at maximal rates around 13-15 mumol/g/hr. The formation of S-(1-chloro-4-nitrophenyl)-glutathione and S-(2,4-dinitrophenyl)-glutathione by the liver decreased the amount of 366-nm light reflected from the liver surface detected with a large-tipped (2 mm) fiberoptic light guide. Initial rates of decrease in reflected light correlated highly with maximal rates of conjugate formation by the liver. Subsequently, micro-light guides were placed on periportal and pericentral regions of the liver lobule. Rates of glutathione adduct formation were calculated from the proportion of the total change in rate of reflected 366-nm light which occurred in each region and the overall rate of product formation by the liver. Changes in the reflectance signal require reduced glutathione (GSH) and were shown to originate from intracellular conjugate formation and not from adducts in the bile canaliculus. Livers from normal rats produced conjugated products from DCNB (100 microM) at maximal rates of 14 and 15 mumol/g/hr in periportal and pericentral regions of the liver lobule, respectively. With CDNB as substrate, changes in reflected light at 366 nm were detected nearly exclusively in periportal regions of the lobule in livers from normal rats. In sharp contrast, CDNB and DCNB were conjugated exclusively in periportal regions of the lobule at rates of 21-22 mumol/g/hr in livers from phenobarbital-treated rats (i.e., the reflectance signal was not altered by these substrates in pericentral areas). When CDNB and DCNB were infused into livers from phenobarbital-treated rats perfused in the retrograde direction, decreases in reflected light at 366 nm were detected initially in pericentral areas followed in about 12 min by changes in periportal regions. Maximal rates of adduct formation in both regions reached 25 mumol/g/hr during perfusion in the retrograde direction. Thus, pericentral regions indeed possess the capacity to conjugate both CDNB and DCNB. When glutathione synthesis was inhibited with L-buthionine sulfoximine treatment (6 mmol/kg), which partially depletes GSH, CDNB was conjugated in both periportal and pericentral regions of the liver lobule in livers from phenobarbital-treated rats.(ABSTRACT TRUNCATED AT 400 WORDS)