Craniosynostosis and risk factors related to thyroid dysfunction.

Thyroid disease is a common problem among women of reproductive age but often goes undiagnosed. Maternal thyroid disease has been associated with increased risk of craniosynostosis. We hypothesized that known risk factors for thyroid disease would be associated with risk of craniosynostosis among women not diagnosed with thyroid disease. Analyses included mothers of 1,067 cases and 8,494 population-based controls who were interviewed for the National Birth Defects Prevention Study. We used multivariable logistic regression to estimate adjusted odds ratios (AOR) and 95% confidence intervals (CI). After excluding women with diagnosed thyroid disease, younger maternal age (AOR 0.7, 95% CI 0.6-0.9, for <25 years versus 25-29), black or other race-ethnicity (AOR 0.3, 95% CI 0.2-0.4 and AOR 0.6, 95% CI 0.4-0.8, respectively, relative to non-Hispanic whites), fertility medications or procedures (AOR 1.5, 95% CI 1.2-2.0), and alcohol consumption (AOR 0.8, 95% CI 0.7-0.9) were associated with risk of craniosynostosis, based on confidence intervals that excluded 1.0. These associations with craniosynostosis are consistent with the direction of their association with thyroid dysfunction (i.e., younger age, black race-ethnicity and alcohol consumption are associated with reduced risk and fertility problems are associated with increased risk of thyroid disease). This study thus provides support for the hypothesis that risk factors associated with thyroid dysfunction are also associated with risk of craniosynostosis. Improved understanding of the potential association between maternal thyroid function and craniosynostosis among offspring is important given that craniosynostosis carries significant morbidity and that thyroid disease is under-diagnosed and potentially modifiable.

Transcriptome correlation analysis identifies two unique craniosynostosis subtypes associated with IRS1 activation.

The discovery of causal mechanisms associated with nonsyndromic craniosynostosis has proven to be a difficult task due to the complex nature of the disease. In this study, differential transcriptome correlation analysis was used to identify two molecularly distinct subtypes of nonsyndromic craniosynostosis, termed subtype A and subtype B. In addition to unique correlation structure, subtype A was also associated with high IGF pathway expression, whereas subtype B was associated with high integrin expression. To identify a pathologic link between altered gene correlation/expression and the disease state, phosphorylation assays were performed on primary osteoblast cell lines derived from cases within subtype A or subtype B, as well as on primary osteoblast cell lines with novel IGF1R variants previously reported by our lab (Cunningham ML, Horst JA, Rieder MJ, Hing AV, Stanaway IB, Park SS, Samudrala R, Speltz ML. Am J Med Genet A 155A: 91-97, 2011). Elevated IRS1 (pan-tyr) and GSK3ß (ser-9) phosphorylation were observed in two novel IGF1R variants with receptor L domain mutations. In subtype A, a hypomineralization phenotype coupled with decreased phosphorylation of IRS1 (ser-312), p38 (thr-180/tyr-182), and p70S6K (thr-412) was observed. In subtype B, decreased phosphorylation of IRS1 (ser-312) as well as increased phosphorylation of Akt (ser-473), GSK3ß (ser-9), IGF1R (tyr-1135/tyr-1136), JNK (thr-183/tyr-187), p70S6K (thr-412), and pRPS6 (ser-235/ser-236) was observed, thus implicating the activation of IRS1-mediated Akt signaling in potentiating craniosynostosis in this subtype. Taken together, these results suggest that despite the stimulation of different pathways, activating phosphorylation patterns for IRS1 were consistent in cell lines from both subtypes and the IGF1R variants, thus implicating a key role for IRS1 in the pathogenesis of nonsyndromic craniosynostosis.

Structure of MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) from Vibrio fischeri in complex with substrate UDP-N-acetylglucosamine and the drug fosfomycin.

The development of new antibiotics is necessitated by the rapid development of resistance to current therapies. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which catalyzes the first committed step of bacterial peptidoglycan biosynthesis, is a prime candidate for therapeutic intervention. MurA is the target of the antibiotic fosfomycin, a natural product produced by Streptomyces. Despite possessing a high degree of sequence conservation with MurA enzymes from fosfomycin-susceptible organisms, recent microbiological studies suggest that MurA from Vibrio fischeri (VfiMurA) may confer fosfomycin resistance via a mechanism that is not yet understood. The crystal structure of VfiMurA in a ternary complex with the substrate UDP-N-acetylglucosamine (UNAG) and fosfomycin has been solved to a resolution of 1.93 Å. Fosfomycin is known to inhibit MurA by covalently binding to a highly conserved cysteine in the active site of the enzyme. A comparison of the title structure with the structure of fosfomycin-susceptible Haemophilus influenzae MurA (PDB entry 2rl2) revealed strikingly similar conformations of the mobile substrate-binding loop and clear electron density for a fosfomycin-cysteine adduct. Based on these results, there are no distinguishing sequence/structural features in VfiMurA that would translate to a diminished sensitivity to fosfomycin. However, VfiMurA is a robust crystallizer and shares high sequence identity with many clinically relevant bacterial pathogens. Thus, it would serve as an ideal system for use in the structure-guided optimization of new antibacterial agents.

Craniofacial and intraoral phenotype of Robinow syndrome forms.

Robinow syndrome (RS) is a rare genetic condition with two inheritance forms, autosomal dominant RS (DRS) and autosomal recessive RS (RRS). The characteristic features of this syndrome overlap in both inheritance forms, which make the clinical differential diagnosis difficult, especially in isolated cases. The objective of this study was to identify differences in the craniofacial and intraoral phenotype of patients with DRS and RRS. The characteristics and frequency of 13 facial and 13 intraoral clinical features associated with both DRS and RRS were assessed by direct dysmorphology examination and using a digital photographic analysis in 12 affected subjects. Although the phenotypic presentation varied and overlapped in the two forms of the syndrome, there were differences in the severity of the craniofacial and intraoral features. The craniofacial dysmorphology of RS was more severe in RRS. Nasal anomalies were the most frequent craniofacial features in both DRS and RRS. In contrast, intraoral features such as wide retromolar ridge, alveolar ridge deformation, malocclusion, dental crowding and hypodontia were more severe in patients with DRS. Overall, facial characteristics appeared less pronounced in adult subjects compared to younger subjects. Craniofacial and intraoral findings are highly variable in RS, with abnormalities of the intraoral structures being more prominent in the DRS form. We propose that the difference in the alveolar ridge deformation pattern and severity of other intraoral characteristics could enhance the differential diagnosis of the two forms of this syndrome.

Three-dimensional head shape quantification for infants with and without deformational plagiocephaly.

OBJECTIVE: The authors developed and tested three-dimensional (3D) indices for quantifying the severity of deformational plagiocephaly (DP). DESIGN: The authors evaluated the extent to which infants with and without DP (as determined by clinic referral and two experts' ratings) could be correctly classified. PARTICIPANTS: Infants aged 4 to 11 months, including 154 with diagnosed DP and 100 infants without a history of DP or other craniofacial condition. After excluding participants with discrepant expert ratings, data from 90 infants with DP and 50 infants without DP were retained. MEASUREMENTS: Two-dimensional (2D) histograms of surface normal vector angles were extracted from 3D mesh data and used to compute the severity scores. OUTCOME MEASURES: Left posterior flattening score (LPFS), right posterior flattening score (RPFS), asymmetry score (AS), absolute asymmetry score (AAS), and an approximation of a previously described 2D measure, the oblique cranial length ratio (aOCLR). Two-dimensional histograms localized the posterior flatness for each participant. ANALYSIS: The authors fit receiver operating characteristic curves and calculated the area under the curves (AUC) to evaluate the relative accuracy of DP classification using the above measures. RESULTS: The AUC statistics were AAS = 91%, LPFS = 97%, RPFS = 91%, AS = 99%, and aOCLR = 79%. CONCLUSION: Novel 3D-based plagiocephaly posterior severity scores provided better sensitivity and specificity in the discrimination of plagiocephalic and typical head shapes than the 2D measurements provided by a close approximation of OCLR. These indices will allow for more precise quantification of the DP phenotype in future studies on the prevalence of this condition, which may lead to improved clinical care.

Embryo lipoproteins and yolk lipovitellin consumption during embryogenesis in Macrobrachium borellii (Crustacea: Palaemonidae).

The prawn Macrobrachium borellii has lecithotrophic eggs with highly-abbreviated development. The major yolk component is lipovitellin (LV), a lipoprotein with 30% lipids (by weight). LV consumption during embryogenesis was followed by ELISA and Western blot analysis using an anti-LV polyclonal antibody. No cross-reacting proteins were observed and LV-like lipoproteins were strongly recognized by the antibody in hemolymph (vitellogenin), yolk (LV) and embryos (LVe), as determined by Western Blot analysis. LV decreased significantly along development from 9.4 to 1.1 microg/mg egg. Consumption rate of LV was slow in early embryogenesis, followed by a rapid utilization in late embryonic stages. Significant LVe amounts were still present at hatching. LV apolipoproteins were selectively degraded during embryo development, being the highest molecular weight subunit the most affected. Comparison among in vitro, in vivo and theoretical proteolysis suggested that trypsin may be involved in LV degradation during late embryogenesis. Embryo lipoprotein (HDLe) synthesis was first detected at stage 6. HDLe shared the same density, MW and subunit composition as adult hemolymph HDL(1) and did not cross-react with LV-like lipoproteins. Though expressed at low concentration, it fulfilled embryo needs for lipid transport among organs.

Predicting neuropsychological development from skull imaging.

Craniosynostosis is a serious and common pediatric disease caused by the premature fusion of sutures of the skull. Although studies have shown an increase in risk for cognitive deficits in patients with isolated craniosynostosis, the causal basis for this association is still unclear. It is hypothesized that an abnormally shaped skull produces a secondary deformation of the brain that results in the disruption of normal neuropsychological development. In this paper, we conduct a comparative analysis of our newly developed shape descriptors in an attempt to understand the impact of skull deformations on neurobehavior. In particular, we show that our scaphocephaly severity indices and symbolic shape signatures are predictive of mental ability and psychomotor functions, respectively, which suggests the possibility that secondary deformation could influence neuro-developmental status.

Differential expression of CYP1A, 2B, and 3A genes in the F344 rat following exposure to a polybrominated diphenyl ether mixture or individual components.

Polybrominated diphenyl ethers (PBDEs), used as flame retardants, have been detected in the environment and in mammalian tissues and fluids. Evidence indicates that PBDE mixtures induce CYPs through aryl hydrocarbon receptor (AhR)-dependent and -independent pathways. The present work has investigated the effects of individual components of a commercial PBDE mixture (DE71) on expression of CYP1A1, a biomarker for activation of the AhR (dioxin-like), and CYP2B and CYP3A, biomarkers for activation of the constitutive androstane and pregnanexreceptors (CAR and PXR), respectively, in the rat. Male F344 rats were dosed orally on three consecutive days with either DE71, PBDE components, 2,2',4,4'-tetraBDE (BDE47), 2,2',4,4',5-pentaBDE (BDE99), 2,2',4,4',5,5'-hexaBDE (BDE153), representative polybrominated dibenzofurans (PBDFs) present in DE71, or reference PCBs. Differential expression of target genes was determined in liver 24 h after the last dose. Quantitative PCR analysis indicated up-regulation of CYP1A1 by DE71; however, the response was weak compared to that for dioxin-like PCB126. Individual PBDE components of DE71 up-regulated CYP1A1 only at the highest administered dose (100 micromol/kg/day). Representative PBDFs efficiently up-regulated CYP1A1; therefore, they, along with other PBDFs and polybrominated dibenzodioxins detected in DE71 and individual PBDE components, may be responsible for most, if not all, dioxin-like properties previously observed for PBDEs. Conversely, PBDEs appear capable of up-regulating CYP2B and CYP3A in rats at doses similar to that for non-dioxin-like PCB153. These results indicate that in vivo PBDE-mediated toxicity would be better categorized by AhR-independent mechanisms, rather than the well-characterized AhR-dependent mechanism associated with exposure to dioxin-like chemicals.

Symbolic shape descriptors for classifying craniosynostosis deformations from skull imaging.

Craniosynostosis is a serious condition of childhood, caused by the early fusion of the sutures of the skull. The resulting abnormal skull development can lead to severe deformities, increased intra-cranial pressure, as well as vision, hearing and breathing problems. In this work we develop a novel approach to accurately classify deformations caused by metopic and isolated sagittal synostosis. Our method combines a novel set of symbolic shape descriptors and off-the-shelf classification tools to model morphological variations that characterize the synostotic skull. We demonstrate the efficacy of our methodology in a series of large-scale classification experiments that contrast the performance of our proposed symbolic descriptors to those of traditional numeric descriptors, such as clinical severity indices, Fourier-based descriptors and cranial image quantifications.

Determination of tamoxifen and metabolites in mouse fetal tissue using nonaqueous capillary electrophoresis.

Tamoxifen (TAM), an antiestrogen, has been approved for use by women at risk for developing hormone-dependent breast cancer. Administration of TAM to pregnant CD-1 mice apparently results in reproductive tract toxicity in female offspring. However, there is little or no data describing potential TAM-induced fetal toxicity to women who may become pregnant while receiving prophylactic TAM treatment. In support of the National Toxicology Program's characterization of reproductive and developmental effects of TAM, the present work describes a capillary electrophoresis (CE)-based analytical technique used for detection of TAM and two major metabolites, N-desmethyltamoxifen (DMT), and 4-hydroxytamoxifen (4-HT) in CD-1 mouse fetal tissue. TAM-derived material was extracted from CD-1 mouse fetuses 2-12 h following TAM administration (100 mg/kg) to dams on gestation day 16. The presence of TAM, DMT, and 4-HT was confirmed in the solvent extracts by nonaqueous CE. The limit of detection of TAM by UV absorption was approximately 675 amol at a signal-to-noise ratio of 2:1. This work demonstrates both transplacental transport of TAM in CD-1 mice and a sensitive analytical technique for detecting low concentrations of TAM and similar compounds in biological tissues.

Hepatic expression of polymerase beta, Ref-1, PCNA, and Bax in WY 14,643-exposed rats and hamsters.

The hepatic levels of three protein markers of oxidative stress, polymerase beta, Ref-1, and PCNA, and of the pro-apoptotic protein, Bax, were quantitated after exposure to WY 14,643 (500 ppm in the feed) for 6 or 34 days in a rodent that is susceptible peroxisome proliferator (PP)-induced liver tumors (the Sprague Dawley rat) and in a rodent that is relatively resistant PP-induced liver tumors (the Syrian hamster). The analysis of detergent-extracted whole liver homogenates by immunoblotting showed a marked increase in the abundance of a 45-kDa variant of polymerase beta immunoreactivity and significant increases in the expression of Ref-1 and PCNA in WY 14,643-exposed rats. In contrast. WY 14,643-exposed hamsters expressed only trace levels of the polymerase beta variant and showed significant decreases in the expression of Ref-1 and PCNA. Long-term WY 14,643 exposure was associated with marked decreases in Bax expression in both species. Dose-response studies in the rat showed that the hepatic expression of the polymerase beta and Ref-1 were significantly increased after 6 days of exposure to WY 14,643 at levels of 5 and 50 ppm, respectively. The analysis of subcellular fractions of rat liver showed that the pathological increases in the levels of polymerase beta, Ref-1, and PCNA were especially prominent in mitochondria-enriched particulate liver subfractions. These results indicate that WY 14,643 exposure is associated with an increase in oxidative stress to the liver and that liver mitochondria are a major target of WY 14,643-associated liver damage. Our data are consistent with the hypothesis that the chronic overexpression of mutagenic or oncogenic effectors like polymerase beta and Ref-1 in a setting of increased hepatocyte proliferation and decreased apoptosis may facilitate peroxisome proliferator-induced hepatocellular carcinoma in the rat.

Another TWIST on Baller-Gerold syndrome.

Baller-Gerold syndrome is characterized by craniosynostosis and preaxial upper limb malformations. Wide heterogeneity exists with regard to the presence of additional anomalies. Most of the 31 reported cases involve other malformations, including cardiac, Central Nervous System (CNS), and urogenital anomalies. Baller-Gerold syndrome is thought to have autosomal recessive inheritance. However, Gripp et al. [1999: Am. J. Med. Genet. 82:170-176] recently provided the first evidence for autosomal dominant inheritance with variable expressivity and severity. A nonsense mutation was found in TWIST, a gene associated with Saethre-Chotzen syndrome (SCS). Here we report on a male Caucasian patient of nonconsanguineous parents, with synostosis of the coronal, metopic, and sagittal sutures, and bilateral radial ray hypoplasia. The patient's small, round ears with prominent crus helices, and cervical anomalies are common features of SCS. The father had very mild features of SCS. We identify direct paternal transmission of a novel missense TWIST mutation in the highly conserved Helix II domain of this bHLH-family gene. This report lends further support to the recent findings by Gripp et al. [1999]. Future TWIST mutational analysis on patients with craniosynostosis and radial ray involvement will shed light on whether Baller-Gerold syndrome should be a distinct entity or some cases should be reclassified as a heterogeneous form of SCS.

Comparison of the mutant frequencies and mutation spectra of three non-genotoxic carcinogens, oxazepam, phenobarbital, and Wyeth 14,643, at the lambdacII locus in Big Blue transgenic mice.

Oxazepam (OX), a widely used benzodiazepine anxiolytic, phenobarbital (PHE), a drug used for convulsive disorders, and Wyeth 14,643 (WY; [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid), a hypolipidemic agent, are all hepatocarcinogenic in B6C3F1 mice. They have been classified as "non-genotoxic" carcinogens since they are non-DNA reactive in in vitro assays and are either negative or weakly positive in Salmonella typhimurium (Ames assay). Male B6C3F1 Big Blue(R) transgenic mice were fed 2500 ppm of OX or PHE or 500 ppm of WY in their diet, while a control group of mice received diet alone for 180 days. The mutant frequency (MF) of cII in the control mice, after correction for clonality, was 6.2 +/- 2.8 x 10(-5). The MF values for mice fed OX, PHE, and WY were 10.0 +/- 3.6 x 10(-5) (P < 0.05), 7.9 +/- 1.3 x 10(-5) (P = 0.1) and 17.4 +/- 4.2 x 10(-5) (P < 0.01), respectively. The mutation spectrum (MS) at cII from the PHE-fed mice was significantly different (P < 0.05) from that of the control mice even though the MF was not, whereas the MS spectra of mice fed OX (P = 0.4) and WY (P = 0.7) were not significantly different. The PHE-derived spectrum differed from the spontaneous spectrum in the lower occurrence of G:C>C:G transversions (17 vs 1.6%) and the higher incidence of A:T>T:A transversions (3.4 vs 9.5%). Prior to correction for clonal expansion, each treated group exhibited a high incidence of frameshift mutations at the homopolymeric run of guanines at bp 179-184 (OX 21%, PHE 21%, WY 16% of the total mutations); this was not the case with the control group (6%). Even after clonal correction, more than 10% of the mutations were frameshifts in the treated mice, while 5% were frameshifts in the control mice. Despite this hypersensitive region of the gene, our findings suggest that the cII locus is less sensitive than the lacI locus to mutation induction by non-DNA reactive carcinogens.

Unilateral aplasia of the middle cranial fossa floor in atypical hemifacial microsomia.

We present the first report of a patient with atypical hemifacial microsomia (HFM) and unilateral aplasia of the floor of the middle cranial fossa, glenoid fossa, and portions of her posterior fossa. The patient also developed a Chiari I malformation with cervical syrinx over a 3-year interval. This case report highlights the critical role of imaging in revealing serious, but clinically occult, structural abnormalities, as well as the evolution in the pathogenetic understanding of HFM.

Inhalation toxicity studies of the alpha,beta-unsaturated ketones: ethyl vinyl ketone.

The National Toxicology Program is conducting a chemical class study to investigate the structure-activity relationships for the toxicity of alpha,beta-unsaturated ketones. Ethyl vinyl ketone (EVK) was selected for study because it is a representative straight-chain aliphatic alpha,beta-unsaturated ketone with extensive use and widespread exposure. Short-term inhalation studies of EVK were conducted to provide toxicity data for comparison with the related alpha,beta-unsaturated ketones 2-cyclohexene-1-one (CHX) and methyl vinyl ketone (MVK). These data will be used in designing chronic toxicity and carcinogenicity studies of these ketones. Male and female F344 rats and B6C3F1 mice were exposed to 0, 2, 4, or 8 ppm EVK 6 h/day, 5 days/wk for 13 wk. The nasal cavity was the major target organ of EVK in both rats and mice. Pathologic findings in both the olfactory and respiratory epithelium were observed. Lesions consisted primarily of olfactory epithelial necrosis, atrophy and regeneration, and/or hyperplasia and squamous metaplasia of the respiratory epithelium. Squamous metaplasia of the respiratory epithelium was present in all rats and mice exposed to 4 and 8 ppm EVK, and these lesions were more severe in rats than in mice. Few systemic effects were observed in rats and mice exposed to EVK. A transient decrease in total leukocytes due to decrements in lymphocyte and monocyte populations was present in male rats after exposure to 8 ppm for 3 and 21 days; however, this effect was not present after exposure for 13 wk. There were no chemical-related effects on micronucleus formation in mice, or on sperm motility and vaginal cytology in either species. EVK, like other alpha,beta-unsaturated ketones, is a reactive, direct-acting gaseous irritant with toxicity limited primarily to the upper respiratory tract.

Differential activation of hepatic NF-kappaB in rats and hamsters by the peroxisome proliferators Wy-14,643, gemfibrozil, and dibutyl phthalate.

Nuclear factor-kappaB (NF-kappaB) is an oxidative stress-activated transcription factor involved in the regulation of cell proliferation and apoptosis. We found previously that the peroxisome proliferator ciprofibrate activates NF-kappaB in the livers of rats and mice. These species are sensitive to the hepatocarcinogenic effects of peroxisome proliferators, whereas other species such as Syrian hamsters are not. In the present study we examined the effects of 3 different peroxisome proliferators on NF-kappaB activation in rats and Syrian hamsters. The peroxisome proliferators Wy-14,643, gemfibrozil, and dibutyl phthalate were administered to animals for 6, 34, or 90 days. NF-kappaB activity was determined using electrophoretic mobility-shift assays and confirmed using supershift assays. Wy-14,643 increased the DNA binding activity of NF-kappaB at all 3 time points in rats and produced the highest activation of the 3 chemicals tested. Gemfibrozil and dibutyl phthalate increased NF-kappaB activation to a lesser extent in rats and not at all times. There were no differences in hepatic NF-kappaB levels between control hamsters and hamsters treated with any of the peroxisome proliferators. This study demonstrates species-specific differences in hepatic NF-kappaB activation by peroxisome proliferators.

Regulation of differentiation to the infective stage of the protozoan parasite Leishmania major by tetrahydrobiopterin.

A critical step in the infectious cycle of Leishmania is the differentiation of parasites within the sand fly vector to the highly infective metacyclic promastigote stage. Here, we establish tetrahydrobiopterin (H4B) levels as an important factor controlling the extent of metacyclogenesis. H4B levels decline substantially during normal development, and genetic or nutritional manipulations showed that low H4B caused elevated metacyclogenesis. Mutants lacking pteridine reductase 1 (PTR1) had low levels of H4B, remained infectious to mice, and induced larger cutaneous lesions (hypervirulence). Thus, the control of pteridine metabolism has relevance to the mechanism of Leishmania differentiation and the limitation of virulence during evolution.

Heterozygous mutations in ANKH, the human ortholog of the mouse progressive ankylosis gene, result in craniometaphyseal dysplasia.

Craniometaphyseal dysplasia (CMD) is a bone dysplasia characterized by overgrowth and sclerosis of the craniofacial bones and abnormal modeling of the metaphyses of the tubular bones. Hyperostosis and sclerosis of the skull may lead to cranial nerve compressions resulting in hearing loss and facial palsy. An autosomal dominant form of the disorder (MIM 123000) was linked to chromosome 5p15.2-p14.1 (ref. 3) within a region harboring the human homolog (ANKH) of the mouse progressive ankylosis (ank) gene. The ANK protein spans the outer cell membrane and shuttles inorganic pyrophosphate (PPi), a major inhibitor of physiologic and pathologic calcification, bone mineralization and bone resorption. Here we carry out mutation analysis of ANKH, revealing six different mutations in eight of nine families. The mutations predict single amino acid substitutions, deletions or insertions. Using a helix prediction program, we propose for the ANK molecule 12 membrane-spanning helices with an alternate inside/out orientation and a central channel permitting the passage of PPi. The mutations occur at highly conserved amino acid residues presumed to be located in the cytosolic portion of the protein. Our results link the PPi channel ANK with bone formation and remodeling.

New models for assessing carcinogenesis: an ongoing process.

Traditionally, the use of rodent models in assessing the carcinogenic potential of chemicals has been expensive and lengthy, and the relevance of the carcinogenic effect to humans is often not fully understood. Today, however, with the rapid advances in molecular biology, genetically altered mice containing genes relevant to humans (e.g. oncogenes, tumor suppressor genes) and reporter genes (e.g. lacI) provide powerful tools for examining specific chemical-gene interactions thereby allowing a better understanding of the mechanisms of carcinogenesis in a shorter period of time. This paper will cover an overview of ongoing validation efforts, followed by examples of studies using several genetically engineered models including the p53def mouse model and the Big Blue transgenic mouse model. Specifically, examples where transgenic models were integrated into the testing program based on specific hypotheses dealing with genetic alterations in cancer genes and reporter genes will be discussed. The examples will highlight possible ways genetically altered mice may be integrated into a comprehensive research and testing strategy and thereby provide an improved estimation of human health risks.