128 Antioxidant Enzymes

14.5 THE ROLE OF DT-DIAPHORASE (DTD) IN BIOREDUCTIVE ACTIVATION OF THE MITOMYCIN CLASS OF ANTITUMOR QUINONES. David Ross, Neil W. Gibson, David Siegel and Peter C. Preusch. Molecular Toxicology and Environmental Health Sciences Program, School of Pharmacy, University of Colorado, Boulder, CO 80309, School of Pharmacy and Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, Department of Chemistry, University of Akron, Akron, OH 44325.

Bioreductive activation of mitomycin C to a species capable of crosslinking DNA is considered critical to its biological activity. We have examined the role of DTD in bioreduction of MC in HT-29 human colon carcinoma cells, a cell with high DTD activity. MC induced DNA interstrand crosslinking and cytotoxicity in HT-29 cells which could be inhibited by dicoumarol, an inhibitor of DTD. MC was found to be a substrate for DTD but metabolism was pH- dependent. Metabolism of MC by HT-29 cell cytosol, purified rat hepatic DTD and purified HT-29 DTD increased as the pH was decreased from 7.8 to 5.8. Metabolism of MC by purified rat hepatic DTD at pH = 5.8 was NADH-dependent and inhibited by dicoumarol or by prior boiling of enzyme. The major metabolite formed during metabolism of MC at acidic pH was characterized as 2,7- diaminomitosene. N-methylmitomycin C (porfiromycin) also demonstrated pH-dependent metabolism by rat hepatic DTD similar to MC. Mitomycins A and B, however, which do not contain a 7- NH2 group were metabolized by DTD at both pH 7.8 and 5.8. MC and porfiromycin, but not mitomycin A or B, could effectively inhibit DTD at pH = 7.8. The inhibition of DTD by MC required NADH and was concentration- and time-dependent. The influence of pH on MC-induced inhibition of DTD may reflect the differential reactivity of the MC-derived quinone methide at different pH values. These data show that substituent effects can modulate metabolism of mitomycins by DTD and that metabolism of MC by DTD may be an important determinant of genotoxicity and cytotoxicity. Supported by NIH CA 51210.

CELLULAR RESISTANCE TO AND METABOLISM OF 4 - H Y D R O X Y - 2 - N O N E N A L : A ROLE FOR G L U T A - THIONE AND GLUTATHIONE TRANSFERASE. D.R. Spitz, S.J. Sullivan, R.R. Malcolm, and R.J. Roberts. Dept. of Pediatrics, U. of Virginia, Charlottesville, VA.

Lipid peroxidation derived aldehydes such as 4- hydroxy-2-nonenal (4HNE) have be hypothesized to be detoxified by conjugation with glutathione (GSH) catalyzed by glutathione transferase enzymes (GST). We have tested this hypothesis using H A - I Chinese hamster fibroblasts and H202-resis tant variants (OC14, OC5) derived from HA-1 (J.Cell.Physiol. 139:592-598).

OC14 and OC5 cells were 2 to 3-fold more resistant than HA-1 to the cytotoxicity of 4HNE as assayed by clonogenic survival. These cells metabolized 2 to 3-fold greater quantities (fmole/cell) of 4HNE and possessed 2 to 3-fold greater total GSH and GST activity (Biochem.J. 267:453-459). Furthermore, GSH was found to react with 4HNE, at pH 6.5 in phosphate buffer , leading to the disappearance of the 223nm UV absorbance of 4HNE. This reaction was completed 3.0 times faster in the presence of GST activity. When H A - I cells were exposed to the reaction mixtures (mixed with media), 4HNE toxicity was reduced in a manner which was dependent on [GSH]. GSH depletion of H A - I , OC14, and OC5 cells (>90%) using buthionine sulfoximine resulted in significantly reduced resistance to 4HNE toxicity as well as significantly reduced 4HNE metabolism. The magnitude of the reduced resistance to 4HNE in GSH depleted cells was similar to the magnitude of the reduced ability to metabolize 4HNE. These results support the hypothesis that GSH and GST provide a biologically significant pathway for the detoxification of aldehydic by-products of lipid peroxidation. (Supported by HL42057, HL33964, and DK38942)

14.6

14.7 CYTOPROTECTIVE EFFECTS OF GLUTATHIONE AND GLUTATHIONE PEROXIDASE ON ENDOTHELIAL CELL INJURY BY LIPID PEROXIDE Ikuo Morita,Hiroshi Ochi,Sei-itsu Murota Section of Physiological Chemistry, Faculty of Dentistry, Tokyo Med. & Dent.Univ. , I-5-45 Yushima, Bunkyo-ku, Tokyo-ll3, Japan

We investigated the role of glutathione and glutathione redox cycle as an antioxidant defence system in endothelial cells against the cellular injury induced by 15-hydroperoxyeicosatetraenoic acid (15-HPETE), an arachidonate lipoxygenase product. Pretreatment of endothelial monolayers with reduced glutathone (GSH) resulted in a marked suppression of 15- HPETE-induced cellu~r injury, which was determined by the J~Cr-release assay. This cytoprotective action of GSH is correlated with the intracellular GSH levels. The degree of 15-HPETE-induced cytotoxicity was varied markedly with various agents capable of modulating the endothelial GSH levels. Interestingly, 15-HPETE treatment did not cause the depletion of intracellular GSH levels, but cause a significant inhibition of GSH peroxidase activity. In addition, ebselen, a mimic agent of GSH peroxidase, revealed a remarkable cytoprotective effect on the 15-HPETE-induced cytotoxicity. These results suggest that GSH and GSH peroxi- dase play a pivotal role in the cyto- protection of endothelial monolayers against the cellular injury induced by lipid peroxide, and also suggest that oxidative modification of LDL in endo- thelial cells is regulated by GSH and GSH peroxidase.

CHANGES IN ANTIOXIDANT ENZYMES IN SKELETAL MUS- CLES OF A NEW DYSTROPHIC MODEL MOUSE (MDX). Kohtaro Asayama, Kazushige Dobashi, Nor ih iko Uchida, Hidemasa Hayashibe, and Kiyohiko Kato. Department of Ped ia t r i cs , Yamanashi Medical Col lege, Tamahocho, Yamanashi 409-38, Japan.

We p rev ious ly demonstrated tha t g l u ta th i one de- pendent a n t i o x i d a n t system was ac t i va ted spec- i f i c a l l y in ske le ta l muscles of both dy/dy mouse and pa t i en ts w i th Duchenne muscular dystrophy. Mdx mouse, which lacks the same gene product as human dyst roph ics , is unique in tha t des t ruc t ion of muscle not accompanied by weakness found at 5 week o ld is complete ly repa i red by 8 week o ld. To determine whether muscle a n t i o x i d a n t enzymes were a f fec ted at e i t h e r of the two stages, h ind- l imb muscles ( rec tus femor is , t i b i a l i s an te r i o r , soleus and extensor d ig i to rum longus) were ob- ta ined from mdx and cont ro l mice. Both copper- z inc and manganese superoxide dismutases (SOD) were assayed by radioimmunoassays. Glu ta th ione peroxidase (GPX) was assayed spec t ropho tomet r i - c a l l y . SODs were low or tended to be low in each muscle of mdx mouse both at 5 and 8 weeks of age. Conversely, GPX a c t i v i t y was increased by 2 - fo lds in each muscle of mdx mouse compared to the con t ro l s . The changes were s i m i l a r to those observed in the previous ser ies of dys t ro - phics. The a c t i v a t i o n of GPX may be an adapt ive response to enhanced ox i da t i ve s t ress secondary to the membrane defec t in dys t roph ic muscles. The a l t e r a t i o n of a n t i o x i d a n t enzymes is a t t r i - buted to muscle regenerat ion ra ther than to the d e s t r u c t i o n .

14.8