(VCCEP) Tier 1 Pilot Submission for BENZENE - Tera

which inhibits CYP 2E1, reduced the increase in tail length by 50% in both cell types. These
results corroborate in vivo findings of Valentine et al. (1996) for absence of increased incidence
of micronuclei in mice lacking CYP 2E1.
6.2.2.5 DNA Adducts
Arfellini et al. (1985) and Mazzullo et al. (1989) reported that benzene binding to nucleic acids
and proteins in rodents was low in most organs, with greatest activity in liver and negligible
effect in lung. Labeling of RNA and protein was an order of magnitude greater than labeling of
DNA. Mazzullo et al (1989) observed a linearity of dose response for benzene (labeled with 3 H
or 14 C) DNA-binding in the rat liver at very low doses (6.23×10 -6 to 6.23×10 -1 mmole/kg), with a
saturation of benzene binding activity at 6.23 mmole/kg, approximately equivalent to 60 ppm. A
single DNA adduct was detected by high-pressure liquid chromatography (HPLC) in benzenetreated
liver DNA, but in the absence of analytical identification, this effect may be the result of
protein contamination. Parodi et al. (1989), in a paper that included Mazzullo as coauthor, used
these adduct data, in conjunction with animal and human data, to propose that benzene toxicity
can best be described by a sigmoid curve, rather than linear, that appears sublinear below 10—
30 ppm and flattens above 50—100 ppm. DNA adducts of phenol, hydroquinone, or
benzoquinone have been reported in vitro (Reddy et al., 1990; Lévay et al., 1993; Bodell et al.,
1993). However, Reddy et al. (1990), using the P 1 enhanced 32 P-postlabeling assay, did not
detect DNA adducts in rat bone marrow, Zymbal gland, liver, or spleen, which are target organs
for benzene-induced toxicity, after daily oral gavage treatments of phenol or a 1:1 mixture of
phenol and hydroquinone. Great care was exercised to separate DNA adducts from
contaminating protein adducts. Reddy et al. (1994) also did not detect adducts in liver, bone
marrow, or mammary glands of mice after daily i.p. injections of 500 mg/kg benzene in corn oil
for 4 days. Subsequently, Pathak et al. (1995), using a range of benzene concentrations from
25 to 880 mg/kg and a variety of injection schedules for periods of up to 14 days, identified one
major and two minor DNA adducts in bone marrow of mice receiving 440 mg/kg, i.p. twice daily
for 3 days. Effects were not seen at single doses up to 880 mg/kg. Co-chromatography
indicated that adducts were identical to those seen after in vitro treatment of bone marrow with
hydroquinone.
Accelerator mass spectrometry (AMS), a nuclear physics technique for detecting cosmogenic
isotopes, has been adapted by Turteltaub and colleagues (1993) to detect DNA adducts
induced by low doses of 14 C-labelled carcinogens, at sensitive levels of 1—10 adducts per 10 12
nucleotides. Creek et al. (1997) examined adduct formation in B6C3F1 male mice given 14 Cbenzene
at doses ranging from 700 pg/kg to 500 mg/kg body weight. Tissues, DNA, and
proteins were analyzed by AMS between 0 and 48 hours post-dose. Liver DNA adducts peaked
at 0.5 hour post-dose, while DNA adducts in bone marrow peaked at 12–24 hours. A larger
percentage of available dose in bone marrow bound to DNA than to liver. Protein adducts were
9- to 43-fold greater than DNA adducts. Both DNA and protein adduct formation were linear
over the dose range. The AMS technique was also used by Mani et al. (1999) to demonstrate
differences in benzene-induced adduct formation between mouse strains and Fischer rats. At a
dose of 5 µg/kg, the magnitude of 14 C-benzene-induced DNA and protein adducts in bone
marrow was greater in B6C3F1 mice > DBA/2 mice > C57Bl/6 mice, and lowest in Fischer rats.
AMS is extremely sensitive to adduction at low exposure levels, but it is expensive and of limited
availability. Further, the occurrence of transient, very low levels of adducts may not be
biologically significant.
Although cytogenetic changes induced by benzene in animals are similar to those reported in
humans, there are no reliable animal models for benzene leukemogenesis. Benzene does
Benzene VCCEP Submission
March 2006
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