|In vitro studies
In the Salmonella assay, acrylonitrile has induced reverse mutations
in strains TA1535 (Lijinsky & Andrews, 1980), TA1535, and TA100 (Zeiger
& Haworth, 1985), but only when hamster or rat S9 was present. Weak
positive results were also reported in several Escherichia coli strains in the absence of metabolic activation (Venitt et al., 1977).
In mammalian cells, acrylonitrile induced hprt mutations in human
lymphoblasts without metabolic activation (Crespi et al., 1985), but not in Chinese hamster V79 cells (Lee & Webber, 1985). In several studies, acrylonitrile was positive at the TK locus in mouse lymphoma L5178 TK+/- cells, either with or without rat S9 (Amacher & Turner, 1985; Lee & Webber, 1985; Myhr et al., 1985; Oberly et al., 1985), and in mouse lymphoma P388F cells with metabolic activation (Anderson
& Cross, 1985). It was also mutagenic at the TK locus in human lymphoblasts
with metabolic activation (Crespi et al., 1985; Recio & Skopek, 1988).
Acrylonitrile induced structural chromosomal aberrations either with or without metabolic activation in Chinese hamster ovary cells (Danford, 1985; Gulati et al., 1985; Natarajan et al., 1985) and without metabolic activation in Chinese hamster lung cells
(Ishidate & Sofuni, 1985). Results for sister chromatid exchanges in
Chinese hamster ovary cells and human lymphocytes both with and without
metabolic activation are mixed (Brat & Williams, 1982; Perocco et al.,
1982; Gulati et al., 1985; Natarajan et al., 1985; Obe et al., 1985; Chang et al., 1990).
Results of in vitro assays for DNA single strand breaks (Bradley, 1985; Lakhanisky & Hendrickx,
1985; Bjorge et al., 1996) and DNA repair (unscheduled DNA synthesis) (Perocco et al., 1982; Glauert et al., 1985; Martin & Campbell, 1985; Probst & Hill, 1985; Williams
et al., 1985; Butterworth et al., 1992) were mixed but more commonly negative in a range of cell types
from rats and humans, with and without activation. Cell transformation
in mouse and hamster embryo cells has also been investigated, with mixed
results (Lawrence & McGregor, 1985; Matthews et al., 1985; Sanner & Rivedal, 1985; Abernethy & Boreiko, 1987; Yuan
& Wong, 1991).
Binding of 2-cyanoethylene oxide to nucleic acids has also been reported in in vitro studies at high concentrations (Hogy & Guengerich, 1986; Solomon &
Segal, 1989; Solomon et al., 1993; Yates et al., 1993, 19946). The formation of acrylonitrile?DNA adducts is increased substantially in the presence of metabolic activation. Under non-activating conditions involving incubation of calf thymus DNA with either acrylonitrile or 2-cyanoethylene oxide in vitro, 2-cyanoethylene oxide alkylates DNA much more readily than acrylonitrile
(Guengerich et al., 1981; Solomon et al., 1984, 1993). Incubation of DNA with 2-cyanoethylene oxide yields 7-(2-oxoethyl)-guanine
(Guengerich et al., 1981; Hogy & Guengerich, 1986; Solomon & Segal, 1989; Solomon et al., 1993; Yates et al., 1993, 1994) as well as other adducts. Compared with studies with rat
liver microsomes, little or no DNA alkylation by acrylonitrile was observed
with rat brain microsomes (Guengerich et al., 1981). DNA alkylation in human liver microsomes was much less than that observed with rat microsomes (Guengerich et al., 1981); although there was no glutathione S-transferase activity in cytosol
preparations from human liver exposed to acrylonitrile, there was some
activity for 2-cyanoethylene oxide (Guengerich et al., 1981).
In vivo studies
@Limitations of the few in vivo studies conducted in which the genotoxicity of acrylonitrile has been investigated preclude definitive conclusions. Data from these studies are also inadequate for characterization of dose-response for comparison between studies or with the cancer bioassays.
Exposure to acrylonitrile in drinking-water resulted in increased
frequency of mutants at the hprt locus in splenic T-cells (Walker &
Walker, 1997). Five female F344 rats were exposed to 0, 33, 100, or 500
mg/litre (0, 8, 21, or 76 mg/kg body weight per day; Health Canada, 1994)
in drinking-water for up to 4 weeks and serially sacrificed throughout
exposure and up to 8 weeks post-exposure. At 4 weeks post-exposure, the
average observed mutant frequency in splenic T-cells was increased in a
dose-related manner (significant at the two highest doses).
Results of a range of assays for structural chromosomal aberrations,
micronuclei in bone marrow, and micronuclei in peripheral blood cells have
been negative or inconclusive, although there was no indication in the
published accounts of three of the four studies that the compound reached
the target site. These include studies in Swiss (Rabello-Gay & Ahmed,
1980), NMRI (Leonard et al., 1981), and C57B1/6 (Sharief et al., 1986) mice and a colLabourative study following exposure by multiple
routes in mice and rats (Morita et al., 1997).
Results of dominant lethal assays were inconclusive in mice (Leonard
et al., 1981) and negative in rats (Working et al., 1987).
In assays for unscheduled DNA synthesis in rats, results were positive only for the liver (Hogy & Guengerich, 1986), equivocal in lung, testes, and gastric tissues (Ahmed et al., 1992a,b; Abdel-Rahman et al., 1994), and, notably, negative in the brain (Hogy & Guengerich, 1986).
In these studies, however, unscheduled DNA synthesis was measured by liquid
scintillation counting to determine [3H]thymidine uptake in the cell population,
which does not discriminate between cells undergoing repair and those that
are replicating. Results for unscheduled DNA synthesis in rat liver and
spermatocytes were negative when [3H]thymidine uptake in individual cells
was determined by autoradiography, which eliminates replicating cells from
the analysis (Butterworth et al., 1992).
Urine from acrylonitrile-exposed rats and mice was also mutagenic
in Salmonella typhimurium following intraperitoneal administration of acrylonitrile
to rats and mice (Lambotte-Vandepaer et al., 1980, 1981). In both species, mutagenic activity occurred without activation. Mutagenic activity was also observed in urine of rats administered acrylonitrile by stomach intubation (Lambotte-Vandepaer et al., 1985). Thiocyanate, hydroxyethylmercapturic acid, and cyanoethylmercapturic acid were not believed to be responsible for urinary mutagenicity.
In in vivo studies in F344 rats administered 50 mg acrylonitrile/kg body weight intraperitoneally,
7-(2-oxoethyl)-guanine adducts were detected in liver (Hogy & Guengerich,
1986). Incorporation of acrylonitrile into hepatic RNA was observed following
intraperitoneal administration to rats (Peter et al., 1983). However, no DNA adducts were detected in the brain, which is the primary target for acrylonitrile-induced tumorigenesis, in this or a subsequent study in which F344 rats received 50 or 100 mg acrylonitrile/kg body weight by subcutaneous injection (Prokopczyk et al., 1988). In contrast, in three studies from one Labouratory, exposure of
SD rats to 46.5 mg [14C]acrylonitrile/kg body weight (50 ΚCi/kg body
weight) resulted in apparent binding of radioactivity to DNA from liver,
stomach, brain (Farooqui & Ahmed, 1983), lung (Ahmed et al., 1992a), and testicles (Ahmed et al., 1992b). In each tissue, there was a rapid decrease in radioactivity of
DNA samples collected up to 72 h following treatment.
It is not clear why acrylonitrile-DNA binding was detected in the
brain in these studies and not in those by Hogy & Guengerich (1986)
or Prokopczyk et al. (1988). The DNA isolation protocols and method for correcting for contaminating
protein in the DNA sample used by Hogy & Guengerich (1986) may have
allowed a more stringent determination of DNA-bound material. Alternatively,
the methods used to achieve greater DNA purity might have caused the loss
of adducts or inhibited the recovery of adducted DNA; more likely, however,
7-oxoethylguanine and cyanoethyl adducts are of little consequence in the
induction of acrylonitrile-induced brain tumours. Indeed, investigation
of the role of cyanohydroxyethylguanine and other adducts in the induction
of these tumours seems warranted.