Recent advances in research on radiofrequency fields and health: 2001-2003.

The widespread use of wireless telecommunications devices, particularly mobile phones, has resulted in increased human exposure to radiofrequency (RF) fields. Although national and international agencies have established safety guidelines for exposure to RF fields, concerns remain about the potential for adverse health outcomes to occur in relation to RF field exposure. The extensive literature on RF fields and health has been reviewed by a number of authorities, including the Royal Society of Canada (1999), the European Commission's Scientific Committee on Toxicity, Ecotoxicity, and the Environment (CSTEE, 2001), the British Medical Association (2001), the Swedish Radiation Protection Authority (Boice & McLaughlin, 2002), and the Health Council of The Netherlands (2002). This report provides an update on recent research results on the potential health risks of RF fields since the publication of the Royal Society of Canada report in 1999 (See Krewski et al., 2001a) and our previous 2001 update (Krewski et al., 2001b), covering the period 2001-2003. The present report examines new data on dosimetry and exposure assessment, biological effects such as enzyme induction, and toxicological effects, including genotoxicity, carcinogenicity, and testicular and reproductive outcomes. Epidemiological studies of mobile phone users and occupationally exposed populations are examined, along with human and animal studies of neurological and behavioral effects. All of the authoritative reviews completed within the last 2 yr have concluded that there is no clear evidence of adverse health effects associated with RF fields. However, following a recent review of nine epidemiological studies of mobile phones and cancer, Kundi et al. (2004) concluded that the possibility of an enhanced cancer risk cannot be excluded. These same reviews support the need for further research to clarify the possible associations between RF fields and adverse health outcomes that have appeared in some reports. The results of the ongoing World Health Organization (WHO) study of mobile phones will provide important new information in this regard.

Low doses of gamma ionizing radiation increase hprt mutant frequencies of TK6 cells without triggering the mutator phenotype pathway

The TK6 lymphoblastoid cell line is known to be mismatch repair (MMR) and p53 proficient. Deficiency in MMR results in a mutator phenotype characterized by microsatellite instability (MSI) and increased hprt mutant frequency (MF). Increased hprt MF is also a biomarker of effect for exposure to ionizing radiation. In order to test if a mutator phenotype could be induced by low doses of gamma ionizing radiation, an hprt cloning assay and a MSI investigation were performed after radiation exposure. The spontaneous MF was 1.6 x 10-6. The groups exposed to 0.2, 0.5 and 1.0 Gy had hprt MFs of 2.3, 3.3 and 2.2 x 10-6, respectively. The spontaneous MSI frequency per allele in non-selected cells was 5.4 x 10-3, as evidenced at the loci D11S35, nm23-H1, D8S135 and p53. MSI frequencies in the groups exposed to 0.2, 0.5 and 1.0 Gy were found to be < 4.7, < 7.7 and < 12 x 10-3, respectively. The frequencies of hprt mutants and MSI found in this study suggest that low doses of ionizing radiation increase hprt mutant frequency without triggering the mutator phenotype pathway.

In vivo transgenic mutation assays.

Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3x10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when scientifically justified. Normally male animals alone are sufficient and normally at least one rapidly proliferating and one slowly proliferating tissue should be sampled. Although, as agreed previously, sequencing data are not normally required, they might provide useful additional information in specific circumstances, mainly to identify and correct for clonal expansion and in some cases to determine a mechanism associated with a positive response.

Conjugated linoleic acid inhibits mutagenesis by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in the prostate of Big Blue rats.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potent mutagen and carcinogen formed at high temperature during the cooking of meat. PhIP induces tumors in the colon and prostate of male rats and in the mammary gland of female rats and has been associated with the etiology of human cancers. We have recently demonstrated that PhIP induces mutations in the prostate in Big Blue transgenic rats. In the current study we have examined the effect of a dietary anti-carcinogen, conjugated linoleic acid (CLA), on PhIP-induced mutagenesis in the prostate. CLA is a mixture of positional and geometric isomers of linoleic acid and has been reported to inhibit various chemical-induced cancers in rodent models. Fifty day old male Big Blue rats were fed a standard diet containing 100 p.p.m. PhIP for 47 days, which induced a mutation frequency of 14.6 x 10(-5) in the prostate, 5.1-fold higher than that of controls. The addition of 1% CLA (w/w) in the diet starting 1 week prior to exposure to PhIP decreased PhIP-induced mutagenesis by 38% (P = 0.03). The predominant class of mutation induced by PhIP is -1 frameshifts involving the loss of G:C base pairs, followed by G:C-->T:A transversions and G:C-->A:T transitions. Addition of CLA to the diet significantly changed the PhIP-induced mutation spectrum; notably, -1 frameshifts and G:C-->A:T transitions were selectively inhibited, suggesting involvement of mismatch repair. This is the first report to show the protective effect of CLA against PhIP-induced mutagenesis in the prostate on both mutation frequency and mutational spectrum. The inhibitory effect of CLA against PhIP-induced mutagenicity suggests a possibility for its application in human chemoprevention studies.

Msh2 deficiency increases the mutation frequency in all parts of the mouse colon.

The Msh2 DNA mismatch repair gene is one of five genes implicated in the pathogenesis of hereditary nonpolyposis colorectal cancer (HNPCC). To address the possible mechanisms of the site-specific occurrence of HNPCC, the effect of Msh2 deficiency on mutations in different parts of the colon was investigated using the BC-1(lacI)/Msh2 double transgenic mouse. Compared to the Msh2(+/+) mice, Msh2(-/-) mice had an 8-9-fold increase of mutation frequency (MF) in the lacI gene from the cecum and the proximal and distal colon. The mutational spectra were also significantly different between Msh2(+/+) and Msh2(-/-) mice, with a significant increase in the frequency of -1 frameshifts and G:C-->A:T base substitutions in the repair-deficient mice. However, in spite of the site-specific predisposition of HNPCC in humans, we found no significant difference in the MF or mutation spectrum between the three parts of the colon in Msh2(+/+), Msh2(+/-), or Msh2(-/-) mice. In addition, 11 independent mutants harboring complex mutations within the lacI gene were recovered in the Msh2(-/-) mice. Interestingly, while the Msh2(+/-) mice displayed an overall MF similar to that observed in the wild-type mice, sequencing revealed a significantly different mutational spectrum between Msh2(+/+) and Msh2(+/-) mice, mainly characterized by an increase in -1 frameshifts. Due to the prevalence of frameshift mutations in HNPCC patients, this haploinsufficiency effect of the Msh2 gene in safeguarding genomic integrity may have important implications for human carrier status.

Coding variants in human double-strand break DNA repair genes.

DNA repair is essential for the maintenance of genomic integrity. Consequently, altered repair capacity may impact individual health in such areas as aging and susceptibility to certain diseases. Defects in some DNA repair genes, for example, have been shown to increase cancer risk, accelerate aging and impair neurological functions. Now that over 115 genes directly involved in human DNA repair have been characterized at the DNA sequence level, the identification of single nucleotide polymorphisms (SNPs) in DNA repair genes is becoming a reality. This information will likely lead to the identification of alleles, or combinations of alleles that affect disease predisposition. This communication summarizes SNPs identified to date in the coding region of 24 human double-strand break repair (DSBR) genes. SNP data for four of these genes were obtained by screening at least 100 individuals in our laboratory. For each SNP, the codon number, amino acid substitution, allele frequency and population information is supplied.

Sex-specific induction of mutations by PhIP in the kidney of male and female rats and its modulation by conjugated linoleic acid.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a recognized mutagen and carcinogen in the colon and prostate of male rats and in the mammary gland of female rats. In the current study, we examined the mutagenicity of PhIP in the kidney of male and female lacI transgenic rats and its modulation by a dietary chemopreventive agent, conjugated linoleic acid (CLA). Sex-specific changes in mutation were observed following PhIP and CLA treatment. Exposure to 100 ppm PhIP through dietary supplementation for 47 days induced a lacI mutation frequency (MF) of 7.7 +/- 0.3 x 10(-5) and 4.7 +/- 1.0 x 10(-5) in the kidney of male and female rats, respectively. The PhIP-induced MFs in the kidney of male and female rats were significantly different from each other and were 300% (P < 0.001) and 60% (P < 0.05) higher than the corresponding controls, respectively. When rats were given CLA along with PhIP, CLA completely inhibited the formation of PhIP-induced mutations in the kidney of female rats, but not in male rats. Comparison of mutational spectra did not detect significant differences between male rats treated with PhIP and PhIP + CLA. However, unlike the -1 frameshifts induced by PhIP in the colon and prostate, which consist primarily of G:C deletions, -1 frameshifts in the kidney involved the loss of both G:C and A:T basepairs. Our data indicate that the kidney of the rats responds in a sex-dependent way to mutagenesis and antimutagenesis by PhIP and CLA. These differences may be related to hormonally regulated induction of P450 enzymes or cell proliferation.

Thymic lymphomas arising in Msh2 deficient mice display a large increase in mutation frequency and an altered mutational spectrum.

Mismatch repair (MMR) genes, such as Msh2, are classified as "mutator" genes, responsible for the microsatellite instability identified in many tumors. In the current study, the mutation frequency and mutational spectrum in thymic lymphoma arising in Msh2 deficient mice are investigated. Thymic lymphoma developed in Msh2-/- background displayed an eight to nine-fold increase in mutation frequency compared to the normal thymi in Msh2 deficient animals. Sequencing demonstrated significantly different mutational spectra between normal thymus tissue and thymic lymphomas in Msh2-/- mice (P=0.02). The tumor mutational spectrum is characterized by an increase in base substitutions occurring at A:T sites, and multiple mutations, as well as a minor increase in -1 frameshifts. We analyzed mutations in different parts of the tumors, and different regional hotspots could be identified. Several hotspot mutations that are a rare event in normal tissues were identified in the tumor tissues. We conclude that thymic lymphomas arising in Msh2 deficient genetic background are hypermutable and the altered mutational spectrum might be an indication of infidelity of DNA replication during tumorigenesis.

Effect of conjugated linoleic acid on the formation of spontaneous and PhIP-induced mutation in the colon and cecum of rats.

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of linoleic acid, has been reported to inhibit chemically induced mammary and colon carcinogenesis in rodents. In a preliminary experiment, we found that CLA significantly reduced the induction of mutations by the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the distal colon in male rats. Here, the chemopreventive properties of CLA were further evaluated by assessing its effect on PhIP-induced mutation and aberrant crypt foci (ACF) in both male and female rats. CLA (1%, w/w) was added to the diet (1) from weaning to 50-day-old, or (2) starting 1 week prior to exposure to PhIP. The 50-day-old Big Blue and F344 rats were then exposed to 100 ppm PhIP for 47 days. No sex differences were observed in mutagenic response to the various treatments in either the distal colon or cecum. The mutation frequency (MF) in the cecum and the distal colon from control animals is 4.3+/-1.3 and 5.3+/-1.4 x 10(-5), respectively showing no statistically significant difference. Administration of PhIP induced a four-fold increase in the MF in the cecum and a seven-fold increase in the distal colon compared to the corresponding controls. Supplementation of the diet with CLA lowered the PhIP-induced MF in the distal colon by 23% (P<0.03), but had no effect in the cecum. The PhIP-induced ACF, determined 9 weeks after the termination of treatment with PhIP, were 0.75 ACF/rat, with 1.7 aberrant crypts /ACF in the colon of male rats, all located in the distal colon. This induction was completely inhibited by the addition of CLA.