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Mechanistic understanding and toxicological significance of CYP1s and CYP19
Cytochrome P450s are drug metabolizing enzymes with broad substrate specificities that are responsible for biological oxidation and reduction reactions. The CYP1 family including CYP1B1 and CYP1C1 are induced by environmental contaminants such as polycyclic aromatic hydrocarbons (PAHs) and the halogenated aromatic hydrocarbons (PCBs and dioxins). CYP19 (aromatase) is responsible for conversion of testosterone to estrogen. Research in our laboratory is studying various aspects of how these genes are regulated to better understand their potential roles in carcinogenesis and endocrine disruption. Fundulus heteroclitus, an estuarine fish, and catfish are being used as model organisms in these studies.

Potential for flavonoids found in natural products to be cancer chemotherapeutics

The hypothesis guiding our research is that certain flavonoids interact with drug metabolizing enzymes in a way that is anticarcinogenic. Thus, some flavonoids could provide cancer chemoprotection. Because CYP1B1 is induced in cancer cells and is involved in activation of estrogen and polycyclic aromatic hydrocarbons (PAHs) to genotoxic metabolites, our goal is to find specific CYP1B1 inhibitors. In the United States, prostate cancer is the second leading cause of cancer death in men, while endometrial cancer is the most common gynecological malignancy in women. Therefore, we are using RL95-2 endometrial and 22Rv1 prostate cancer cells to investigate these hypotheses.

Use of in vitro bioassay to assess sediment and water samples for estrogenic and CYP1A-mediated activities
In vitro tests are being increasingly used as screening tools in risk assessments. In vitro bioassays have several advantages over in vivo approaches for environmental monitoring. Typically they offer a cheaper and more rapid way to screen large numbers of samples with high statistical power for ability to cause a biological response indicative of a particular mechanism of action. We use the H4IIE rat hepatoma bioassay to screen for CYP1A-mediated EROD induction by sediment extracts, and the yeast estrogen screen (YES assay) to assess potential of environmental samples to activate the human estrogen receptor. We have used these methods to screen samples from Mobile Bay, the Miami Canal and Biscayne Bay Florida, and most recently to track coastal recovery following hurricane Katrina. (collaboration with M. Slattery, UM and P. Gardinali, FIU).

Alcohol use disorders: involvement of alcohol metabolizing enzyme genes.

One of the most common public health problems in the United States is the disorders associated with alcohol drinking. Our approach to the problem is associated with the identification and characterization of specific genes involved in fetal alcohol syndrome (FAS), a lifelong completely preventable set of physical, mental and neurobehavioral birth defects associated with maternal alcohol consumption during pregnancy. Japanese medaka (Oryzias latipes) embryos serve as models in this research.  Success has been achieved in characterizing several alcohol metabolizing enzyme genes in this fish and identified aldehyde dehydrogenase (ALDH) is the primary gene whose expression is  attenuated by alcohol.  We are also screening drugs suitable in the treatment and prevention of FAS in humans, and have initiated screening with natural compounds like vitamins /natural products.

Chirality influence on biological activity
Sulfoxidation of thioether-containing organophosphate (OP) insecticides significantly enhances the potency of cholinesterase inhibition.  Earlier reports have demonstrated that recombinant flavin-containing monooxygenase 1 (FMO1) catalyzes the oxidation of the OP pesticide fenthion to (+)-fenthion sulfoxide in a stereoselective fashion.  In order to elucidate the absolute configuration of the sulfoxide metabolite produced, we established an efficient synthesis of both enantiomers of fenthion sulfoxide (2), which were subsequently transformed to chiral fenoxon sulfoxides (4) using a two-step protocol.   The use of chiral oxidants, namely, N-(phenylsulfonyl)(3,3-dichlorocamphoryl) oxaziridines, afforded enantioenriched fenthion sulfoxides with high ee (> 82%) from the parent sulfide.  Single recrystallizations afforded chiral fenthion sulfoxides with > 99% ee, measured by chiral HPLC analysis.  The absolute configuration of the (+)-sulfoxide generated from fenthion metabolism by FMO1 was determined to be (R)-(+)-fenthion sulfoxide, confirmed by X-ray crystallographic analysis.  Evaluation of the inhibition of purified eel acetylcholinesterase (AChE) by fenthion and the individual fenthion and fenoxon sulfoxide enantiomers revealed stereoselective inhibition by R-(+)-fenoxon sulfoxide.  Although the enantiomers of fenthion sulfoxide also inhibited AChE more than parent fenthion and fenoxon, no stereoselective effects were observed.  Consequently, FMO1 may not only activate fenthion through sulfoxidation to a more toxic metabolite, but subsequent oxidative desulfuration may lead to (R)-(+)- fenoxon sulfoxide formation and further bioactivation.
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