THE EVALUATION OF THE CARCINOGENICITY OF ASPARTAME
The use of genetically altered or transgenic models for cancer research is a result of years of research into the genetic mech- anisms for cancer development. It is now generally accepted that there are at least two major classes of genes involved in carcinogenesis, tumor suppressor genes and oncogenes. Loss of function (inactivation) of tumor suppressor genes or gain of function (activation) of oncogenes has been identified in the majority of human cancers. Advances in molecular biol- ogy have provided tools to inactivate or insert these specific genes.
According to the Knudson two-hit hypothesis (Knudson et al., 1975; Knudson, 1996) of carcinogenesis, genetically altered models should be more susceptible to induction of tumors by genotoxic carcinogens because they already carry one muta- tion in their germline. Thus, these models should represent a more sensitive and rapid detection system for genotoxic car- cinogens than the classic 2-year chronic rodent bioassays us- ing wild-type animals. The most commonly used models are
the p53+/−, Tg.AC, TgrasH2 and the XPA−/−(Jacobson-Kram
et al., 2004). Only the three models that have been used for as- sessment of aspartame carcinogenicity are discussed here. These
are the p53+/−mouse, the Tg.AC mouse, and the Cdkn2a defi-
Description of Models p53 Model
The heterozygous p53 mouse model was designed based on extensive evidence that p53 is a commonly mutated tumor sup- pressor gene in a wide variety of human tumors. The function of a tumor suppressor gene is to “guard the genome” and expression of the wild-type gene product is necessary for proper function and suppression of oncogenic events. Loss of functional protein, either due to mutations in the protein or loss of the gene, results in greatly increased susceptibility to tumor development. Thus mice that are completely deficient in p53 because of homozygous
null allele (−/−) will spontaneously develop tumors (primar-
ily lymphomas and sarcomas) within the first 3 to 6 months of
life. The heterozygous p53+/− mouse model has one copy of
the functional wild type, and one null allele, which is not tran- scribed or translated, resulting in a lower level of p53 protein (French et al., 2001). These mice develop tumors spontaneously as well, but at a much lower incidence and longer latency time (approximately 9 months) as compared to the homozygous null mice.
As heterozygous p53 mice already carry one mutation in their germline, they should be more susceptible to induction of tumors by genotoxic carcinogens. Thus one explanation for the increased sensitivity of heterozygous p53 model to tumor development is that the carcinogen causes mutation or loss of the second copy of p53, completely depleting the animal of the tumor suppressor gene product. Alternatively, even without a di- rect hit on the second p53 gene, the lower amount of p53 protein may cause an acceleration of tumor development initiated in the other gene. This is termed a gene dosage effect (Venkatacha- lam et al., 2001). The limitation of this model is that it does not detect nongenotoxic carcinogens in a short (6-month) protocol. A longer 9-month protocol, as was used in the NTP study, was recommended.
Storer et al. (2001) reviewed the available data from carcino-
genicity studies with the p53+/− transgenic mouse model to
assess its usefulness as a short-term carcinogenicity assay. A to- tal of 48 different compounds had been tested, some in multiple studies. All studies were conducted using a standardized proto-
col approved by ILSI. In all cases, the mice were p53+/−het-
erozygous mice; however, the background strain varied, includ- ing C3H, CBA, MIH, and C57BL6. The duration of the study was 26 weeks. Overall, 42 of the 48 compounds gave results that were concordant with expectations. In general, nongeno- toxic compounds were negative, and most genotoxic carcino-
gens were positive. p-Cresidine gave positive results in 18/19
studies for bladder cancer.
In addition to use in studies for regulatory carcinogenicity as-
sessments, the p53+/−transgenic model has been widely used
in the cancer research community to assess dietary agents, exer- cise and pharmaceutical chemopreventive agents for their pro- motional or inhibitory effects on cancer development. Although a review of these studies is beyond the scope of this monograph,
Hursting et al. (2004) provides an excellent example of use of p53-deficient mice models by the National Cancer Institute to investigate diet–gene interactions.
Tg.AC Model
This strain contains the v-Ha-rasoncogene, which has been
activated with two mutations. The expression of the product of the gene is regulated by a promoter and is not normally expressed in adult tissues. Exposure to UV light, specific chemicals, and full-thickness wounding induces expression of the transgene, which is necessary to invoke tumor development (Tennant et al., 1995, 1998). The model can detect both genotoxic and nongeno- toxic carcinogens, but was not positive for chemicals that have shown a strain-specific or species-specific response in a 2-year bioassay (Tennant et al., 2001). Thus, the initial concerns that this model would have many false positives have proven to be in- valid. In reviews of over 40 studies with this model, the Tg.AC model was more prone to false negatives (Eastin et al., 2001; Pritchard et al., 2003).
This model is most widely accepted for testing of dermal applications as the skin of this genetically-altered mouse acts as if already initiated with a carcinogen, i.e., skin papillomas will develop within 12 weeks following the application of classic promoter compounds (such as TPA) without a prior application of a carcinogen (Jacobson-Kram et al., 2004).
The oral route of administration of carcinogens also generates tumors, including squamous cell papillomas and carcinomas of the forestomach (Tennant et al., 1998). Although this model has also been used with oral exposure, it has been more fully evaluated with dermal applications.
Cdkn2a-Deficient Model
This model is not widely used, and therefore has not been as well characterized or evaluated as the two already described. It was developed in 1996 by Serrano et al. (1996), and is based on the frequent detection of mutations and deletions in the Cdkn2a gene in a wide variety of tumors. Of special importance is the evidence that genetic alterations in this gene play an important role in human brain cancers (Ohgaki et al., 2004), making use of this model particularly relevant for suspected brain carcinogens. The role of the Cdkn2a gene is complex. This gene codes for a number of proteins that function as cyclin-dependent ki- nase (CDK) inhibitors. Depending on the exon, open reading frame, and polyadenylation sites used, the resulting Cdkn2a gene
transcript will be the p16Ink4avariant or the p19Arfvariant. The
p16Ink4avariant codes for a protein (called p16) that functions
to inhibit CDK4, and the p19Arfvariant protein product (called
p19) functions to stabilize p53 protein. The presence of p16 re- sults in inhibition of CDK4, preventing phosphorylation of pRb, which in turn blocks the transition from G1 to the S phase. The presence of p19 results in sequestration of the protein Mdm2,
FIG. 1. The role of p16 in the G1-S phase transition of the cell cycle (http://www.biocarta.com/pathfiles/m cellcyclePathway. asp).
which prevents Mdm2 from binding to p53 and targeting p53 for degradation. Thus, p53 is available to prevent the G2 to M phase transition. Thus both proteins from the Cdkn2a gene play a critical role in cell cycle regulation (Lubet et al., 2005). The pathways illustrating the role of these proteins in cell cycle reg-
ulation from Biocarta44are shown in Figure 1 and Figure 2.
The net result of these actions is that the Cdkn2a gene is acting as a tumor suppressor gene, and loss of the functions of this gene, as in the transgenic Cdkn2a deficient model, increases susceptibility to tumor formation. This model is also referred to
as theINK4a/ARFmodel because of the two variants described
above that are affected. Although the model has not been widely used in studies assessing carcinogenicity of chemicals, it is a well-established model for cancer research due to the prevalence of mutations in this locus in commonly occurring cancers such as breast cancer (D’Amico et al., 2003).
APPENDIX V: USE OF TRANSGENIC MODELS IN