evidence of an ARE in rat UGT1A6 but were able to show that oltipraz, which
normally activates AREs was acting through an XRE (Auyeung, 2003)
indicating that oltipraz is a mixed ARE/XRE agonist like beta-naphthoflavone
(Kohle and Bock, 2006). Munzel et al. reported that there were three ARE-
like motifs in human 1A6 including one very close to the XRE (Munzel,
2003).
3.1.5 Pharmacogenetics
Genetic polymorphisms have been identified in all of the UGT enzymes that
have been extensively evaluated. The most well-known polymorphism occurs
in UGT1A1. In Caucasians, a TA insertion into a TATA box (UGT1A1^28 )
results in lowered expression of the enzyme and mild hyperbilirubinemia
(Gilbert’s syndrome) in most subjects who carry two variant alleles. The
frequency of the variant in Caucasians is approximately 15%. Neonates who
are homozygous UGT1A1^ 28/^28 have a high rate of neonatal jaundice
requiring light therapy. Gilbert’s patients also experience a significantly higher
rate of neutropenia from irinotecan, and the FDA has approved a genotyping
test for patients taking this anticancer medication. Incidence of Grade 2 or
greater hyberbilirubinemia is significantly higher in Gilbert’s patients taking
atazanavir or indinavir, two HIV protease inhibitors known to inhibit
UGT1A1 in vitro. Polymorphisms in the UGTs and their relationship to
cancer incidence or treatment has been recently reviewed by the authors (Nagar
and Remmel, 2006). Guillemette published an extensive review on the
pharmacogenetics of the UGTs in 2003, but this is a highly active area of
research that is rapidly maturing. Information on polymorphisms for the
UGT1A and UGT2B families can be found on the UGT Web site with
appropriate references at http://galien.pha.ulaval.ca/labocg/alleles/alleles.html.
3.1.6 Experimental Considerations
3.1.6.1 Microsomal Incubation Conditions Incubations in animal or human
liver microsomes are the most common way to determine activity in the
presence of added substrate, UDPGA, Mg2+, and a buffer. As there is no
method available to directly determine enzyme concentration, the incubations
are standardized by addition of the same amount of protein (typically 0.25–
1.0 mg protein/1 mL) after determination of linearity of product formation
with respect to protein concentration and time. In general, the enzyme is stable
up to 45 min to 1 h. Because of the location of the enzyme, a portion of
the microsomal vesicle will be obtained in the normal configuration with the
enzyme active site entrapped within the vesicle. Since UDPGA must
have access to the active site, and the UDPGA influx transporter is not
operative without ATP, it may be necessary to ‘‘activate’’ or ‘‘remove latency’’
of the enzyme. In the past this has been achieved by a variety of methods, but
most commonly by addition of detergents such as Brij 58, Lubrol, or Triton X
56 CONJUGATIVE METABOLISM OF DRUGS