Collaborators  University of Washington, Seattle Clement Furlong, PhD Rebecca Richter University of CA, Berkeley Brenda Eskenazi, PhD Asa Bradman, PhD Kim Harley, PhD Karen Huen, PhD Student Nishat Shaikh, MS Student Ray Lai

Functional genomics is an emerging discipline focusing on determining gene function. It has grown out of mapping and sequencing the genome to begin to discover how individual responses to exogenous substances vary according to inter-and intra-individual differences.

In this study, we are attempting to determine the role of the enzyme paraoxonase (PON1) in the developmental toxicity and neurotoxicity of organophosphorus (OP) insecticides. The neurotoxic effects of organophosphate (OP) pesticides are due mainly to inhibition of acetylcholinesterase (AChE), the enzyme that hydrolyzes acetylcholine (ACh).  Inhibition of AChE causes continued stimulation of neurons due to accumulation of ACh and thus, suppression of neurotransmission to organs. The primary defense mechanism in humans, however, is the enzyme paraoxonase 1 (PON1), which detoxifies various OP pesticides and prevents inhibition of ChE. Many genetic polymorphisms of the PON1 gene have been identified and several have been reported to affect either enzyme activity or enzyme levels. The detoxifying efficiency of PON1192 depends on the gene polymorphisms (QQ <QR<RR). PON1 status, or activity, is a function of genotype plus phenotype. Several promoter region polymorphisms including –108C>T are associated with variation in levels of enzyme.

Using a high throughput 2-substrate enzyme-kinetic analysis (Richter & Furlong, 1999), rates of diazoxon on hydrolysis are plotted against rates of paraoxon kinetic analysis. The advantages of this method are (1) it uses less than 10 µl (in triplicate) of plasma;
(2) genotype is established simultaneously with activity (expression); (3) it will detect information that PCR will not, for example, an allele with a defective promoter element; (4) it will identify a 14-fold variation in individuals with the same genotype; and (5) it opens up the opportunity for additional functional genomics.

New PON1 Polymorphisms

Genetic polymorphisms of PON1 including the single nucleotide polymorphism (SNP) at position 192 and other SNPs along promoter and coding regions of the gene affect detoxification efficiency as well as enzyme levels. Research has identified over 200 genetic variants in the coding and non coding regions of the PON1 gene.  However, their functional importance is unknown.
Other genes that may be of interest include   additional members of the PON family, PON2 and PON3, which also reside on chromosome 7.  Genes involved in the P450 system are also involved in OP metabolism and therefore may also prove to play an important role. 

Future Directions

We will examine the relationship of host factors (age, gender, and ethnicity) and PON1 status.
Specifically, we will:
•Determine PON1 gene frequencies and phenotype in a cohort of Latina pregnant women and their children (CHAMACOS)
•Determine whether maternal factors, including genotype and PON1 expression, affect children’s PON1 status
•Describe the ontogenetic development of PON1 activity in children ages 0-24 months
•Examine whether PON1 status modifies the relationship of exposure with growth and neurodevelopment in children.
•Determine whether there are ethnic and gender differences in PON1 gene frequencies and phenotype.
•Investigate the role of promoter and other polymorphisms in PON1 status in humans