A thesis sUbmi t t e d to t he School of Grad u a t e Studies i n partial fU lfillment of the requ irements for the degree of
The microbicidal efficacies of two anionic surfactants, sodium lauryl sulfate (SLS) and n-lauroylsarcosine (LS), were evaluated in cultured cells and in a murine model of herpes simplex type 2 (HSV-2) intravaginal infection. In vitro studies showed that SLS and LS were potent inhibitors of the infectivity of HSV-2 strain 333. The concentrations of SLS which inhibit viral infectivity by 50% (50% inhibitory dose) and 90% (90% inhibitory dose) were 32.67 and 46.53 M, respectively, whereas the corresponding values for LS were 141.76 and 225.30 M. In addition, intravaginal pretreatment of mice with thermoreversible gel formulations containing 2.5% SLS or 2.5% LS prior to the inoculation of HSV-2 strain 333 completely prevented the development of genital herpetic lesions and the lethality associated with infection. Of prime interest, no infectious virus could be detected in mouse vaginal mucosa. Both formulations still provided significant protection when viral challenge was delayed until 1 h after pretreatment. Finally, intravaginal application of gel formulations containing 2.5% SLS or 2.5% LS once daily for 14 days to rabbits did not induce significant irritations to the genital mucosa, as demonstrated from macroscopic and histopathologic examinations. These results suggest that thermoreversible gel formulations containing SLS or LS could represent potent and safe topical microbicides for the prevention of HSV-2 and possibly other sexually transmitted pathogens, including human immunodeficiency virus.
The role of cytochrome P-450 in me stimulation of lipid peroxidation induccd by thc mycotoxin ochratoxin A (OTA) has been investigated. Purified cytochrome P-450 (liB I) could effectively replace EDTA in stimulating lipid pefOxidation in a reconstituted system l'Onsisling of phospholipid vesicles, NADPH-cYlochrome P-450rcduc!ase, FeJ+, EDTA and NADPll, suggesting that it could mediate the transfer of electrons from NADPH to foc3,. Microsomcs isolated from liven of cobalt protoporphyrin IX-treated rllts (in which cytochrome P-450 Wll" depletedj underwent OTA-dependent lipid peroxidation much more slowly tlmn control microsomes.The role of cytochrome P-450 in OTA met.1bolism was also investigated. To dClermine which cytochrome P·450 isoforms are involved in the metabolism ofOTA, we used different cytochrome P-450 inducers to induce the major isofonns of cytochrome 1'-450 in lhe rm liver.Microsomes from these livers were used to investigate their effect on OTA metabolism.Pretreatment ofmts with pregnenolone -16c(·carbonitrile (peN), phenobarbit.ll (PB), 3-ll1cthyl cholanthrene (3MC), and isosafrole (ISF) greatly induced 4(R)-4-0H-OTA fonmllion; 4(5)-4-011-OTA fonnation was also induced after pretreatment with PB, PCN, 3MC and ISr:. INII pretreaunem primarily induced me 4(5) isomer formation, The fonnation of the 4(R) .md 4 (5) isomeri showed significant differences with respect to pH optima, effect of antioKidants ami iron chelators, The 4(R) isomer fonnation showed a pH optimum of 6.0 using microsornes from rJIs treated with 3MC and ISF, and 6.5 using mierosomes from rats treated with PI] :tnd peN and Wi!S not inhibited by antioxidants or iron chelators. In contmst, both the 4(5) isomer fonmllion and lipid pcroxidation showed a pH optimum of 7.0 -7,5 and both activities were highly sensitive to inhibition by antioxidants and iron ehelators. Lipid peroxides were no: involved in the 4(5) isomer fonnation since addition of linoleic acid hydroperoxidc to microsomcs did not give rise 10 the 4(5) isomer, Cytochrome P-450 appeared to be essenlial since other hemoprolcins such as horseradish peroxidase and hemoglobin were ineffective in metabolizing OTA. Microsomes from rats pretreated with Co-protoporphyrin IX resulted in no metabolil,rn of ochratoxin A. 7-Ethoxy-and 7-pentoxyresorufin assays showed specificity towards cytochromes P-450 induced by 3MC (fA I/lA2) and PB (IIBI) respectively. Also, metyrapone (inhibitor of cytochrome P·450 IIBt) preferentially inhibited OTA mel1bolism by microsomes from rats treated with PB, and I(-naphthoflavone (inhibitor of cytochrome P-450 IAI/lA2) preferentially inhibited OTA metabolism by microwmes from 3MC and ISF treated rats_ Monoclonal antibodies (MAbs) 1-7-1 (against P-450 lA 1/lA2) and 2-66-3 (against P-450 liB I) showed preferential inhibition ofOTA metabolism by microsomes from 3MC and PB treated rats respectively.Excretion of renal enzymes in urine is a sensitive non-invasive index of renal dam:lge.Therefore. we examined the effect of cytochrome P-450 induction on the...
The role of cytochrome P-450 in the stimulation of lipid peroxidation by the nephrotoxic mycotoxin ochratoxin A has been investigated. Ochratoxin A was previously shown to markedly stimulate lipid peroxidation in a reconstituted system consisting of phospholipid vesicles, NADPH-cytochrome P-450 reductase, Fe3+, ethylenediaminetetraacetic acid (EDTA), and reduced nicotinamide adenine dinucleotide phosphate (NADPH). We now show that purified cytochrome P-450IIB1 could effectively replace EDTA in stimulating lipid peroxidation suggesting that it could mediate the transfer of electrons from NADPH to Fe3+. Cobalt protoporphyrin is known to cause an extensive and long-lasting depletion of hepatic cytochrome P-450 in rats, and it has been used to evaluate the role of hepatic cytochrome P-450 in xenobiotic metabolism and toxicity. We have observed that microsomes isolated from livers of cobalt protoporphyrin-pretreated rats underwent ochratoxin A-dependent lipid peroxidation much more slowly than control microsomes. Also, the level of ethane exhaled (an index of in vivo lipid peroxidation) on ochratoxin A administration was much lower in cobalt protoporphyrin-pretreated rats than in control rats. Taken together, these results provide evidence for the stimulatory role of cytochrome P-450 in ochratoxin A-induced lipid peroxidation in a reconstituted system and strongly implicate its role in microsomal and in vivo ochratoxin A-induced lipid peroxidation.
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