It recently has been recognized that men develop colonic adenomas and carcinomas at an earlier age and at a higher rate than women. In the Apc Pirc/+ (Pirc) rat model of early colonic cancer, this sex susceptibility was recapitulated, with male Pirc rats developing twice as many adenomas as females. Analysis of large datasets revealed that the Apc Min/+ mouse also shows enhanced male susceptibility to adenomagenesis, but only in the colon. In addition, WT mice treated with injections of the carcinogen azoxymethane (AOM) showed increased numbers of colonic adenomas in males. The mechanism underlying these observations was investigated by manipulation of hormonal status. The preponderance of colonic adenomas in the Pirc rat model allowed a statistically significant investigation in vivo of the mechanism of sex hormone action on the development of colonic adenomas. Females depleted of endogenous hormones by ovariectomy did not exhibit a change in prevalence of adenomas, nor was any effect observed with replacement of one or a combination of female hormones. In contrast, depletion of male hormones by orchidectomy (castration) markedly protected the Pirc rat from adenoma development, whereas supplementation with testosterone reversed that effect. These observations were recapitulated in the AOM mouse model. Androgen receptor was undetectable in the colon or adenomas, making it likely that testosterone acts indirectly on the tumor lineage. Our findings suggest that indirect tumor-promoting effects of testosterone likely explain the disparity between the sexes in the development of colonic adenomas.colon cancer | animal models | androgens | estrogens | intestinal regionality E pidemiologic studies have identified a number of factors that influence the risk of sporadic adenomas and colorectal cancer (CRC). Age, familial predisposition, racial background, diet, physical activity, obesity and the metabolic syndrome, smoking, and heavy alcohol use are all established risk factors for the development of CRC. In addition, the risk of CRC also shows sexual dimorphism, with a lower incidence and delayed onset in women (1, 2). Colonoscopic screening of asymptomatic individuals has corroborated male sex as a risk factor for the development of both adenomas and CRC in all age groups (3, 4); however, whether this disparity depends on protective factors in women, tumor-promoting factors in males, or both is unknown.A protective role of female hormones against the development of frank CRC is suggested by data from the Women's Health Initiative (WHI). In the WHI, two large randomized controlled trials examined the effects of hormonal replacement therapy on postmenopausal women over a 5-y period, using CRC development as one of the endpoints. The first study showed that combined treatment with both equine estrogen (E2) and medroxyprogesterone acetate (MPA) substantially reduced the risk of colorectal cancer compared with placebo (odds ratio, 0.63) after a 5-y follow-up (5). However, protection was not found in a second randomized cont...

Mutant mice exhibiting heritable hyperphenylalaninemia have been isolated after ethylnitrosourea mutagenesis of the germ line. We describe one mutant pedigree in which phenylalanine hydroxylase activity is severely deficient in homozygotes and reduced in heterozygotes while other biochemical components of phenylalanine catabolism are normal. In homozygotes, injection of phenylalanine causes severe hyperphenylalaninemia and urinary excretion of phenylketones but not hypertyrosinemia. Severe chronic hyperphenylalaninemia can be produced when mutant homozygotes are given phenylalanine in their drinking water. Genetic mapping has localized the mutation to murine chromosome 10 at-or near the Pah locus, the structural gene for phenylalanine hydroxylase. This mutant provides a useful genetic animal model affected in the same enzyme as in human phenylketonuria.Disorders ofphenylalanine catabolism, resulting in phenylketonuria (PKU) and hyperphenylalaninemia (HPH), were among the first heritable errors of metabolism discovered in the human (1). The rate-limiting step in mammalian phenylalanine catabolism is hydroxylation to produce tyrosine. This reaction, catalyzed by phenylalanine hydroxylase (PAH) (2), requires the reduced pteridine cofactor tetrahydrobiopterin (3), which is synthesized from GTP (4) through a number of intermediates and is maintained in its reduced form by quinonoid dihydropteridine reductase (q-DHPR) (5). Mutations reducing the activity of PAH, q-DHPR, or the enzymes involved in tetrahydrobiopterin synthesis result in HPH because of a block in phenylalanine hydroxylation (6). In humans, PKU is defined as a condition resulting from mutations that abolish or severely reduce PAH activity (7). Other defects in phenylalanine catabolism are termed HPH. Extensive research has been undertaken to characterize these disorders (early work is reviewed in refs. 8 and 9 with recent summations in refs. 10 and 11). Laboratory mice with defined PKU and HPH mutations would be helpful in evaluating features of these diseases by permitting investigations not acceptable with human subjects. To produce such mutants we have used the alkylating agent N-ethyl-N-nitrosourea, which induces mutations in the mouse germ line at a frequency near 10-3 per locus (12). We refer to all mutants with deficiencies in phenylalanine catabolism by their common phenotype, HPH. We have screened the progeny of 347 gametes and have isolated four mutant pedigrees exhibiting the HPH phenotype. One of these, HPH-1, was detected by its neonatal HPH phenotype and is deficient in GTP cyclohydrolase activity (13)(14)(15). The others were detected by their impaired ability to clear a phenylalanine challenge (described in ref. 13). One of these, HPH-5, we now report to be deficient in PAH. MATERIALS AND METHODSBiochemical Determinations. Liver homogenates were prepared for PAH assay as described in ref. 16 methyltetrahydropterin, and the enzyme extract. The background rates of NADH oxidation were determined in the absence ofphenylalanine and ind...

Frog rod outer segments freshly detached from dark-adapted retinas contain approximately 1-2 molecules of guanosine 3',5'-cyclic monophosphate (cyclic GMP) for every 100 molecules of visual pigment present. This cyclic GMP decays to 5'-GMP, and the conversion is accelerated upon illumination of the outer segments. Bleaching one rhodopsin molecule can lead to the hydrolysis of 1,000-2,000 molecules of cyclic GMP within 100-300 ms. The decline in cyclic GMP concentration becomes larger as illumination increases, and varies with the logarithm of light intensity at levels which bleach between 5 X 10(2) and 5 X 10(5) rhodopsin molecules per outer segment-second. Light suppression of plasma membrane permeability, assayed in vitro as light suppression of outer segment swelling in a modified Ringer's solution, occurs over this same range of light intensity. The correlation between cyclic GMP and permeability or swelling is maintained in the presence of two pharmacological perturbations: papaverine, a phosphodiesterase inhibitor, increases both cyclic GMP levels and the dark permeability of the plasma membrane; and beta,gamma-methylene ATP increases the effectiveness of light in suppressing both permeability and cyclic GMP levels.

The proximal region of mouse chromosome 17 contains many genes affecting embryonic development, germ cell differentiation, and the immune system. Although the study of natural variation, including t haplotypes, has yielded some information about the function of these genes, spontaneous variants often exhibit manifold genetic effects and are generally not carried on inbred backgrounds. To clearly connect phenotypes with the actions of individual genes, mutants in which genes are altered singly are needed. Therefore, we used a highly efficient point mutagen, N-ethyl-Nnitrosourea, in combination with classical breeding schemes to induce and identify recessive lethal mutations in the tregion. Of 350 mutagenized gametes examined, at least 10 independent recessive embryonic lethal mutations have been identified; an additional two are perinatal lethals. A spontaneous brachyury mutation, TWU, arose on a genetic background that permits high-resolution mapping of the induced recessive mutations against cloned DNA sequences from the t region. One lethal mutation is an allele at the quaking locus. The multiple alleles of quaking and the feasibility of high-resolution mapping permit investigation of the pleiotropic action of this locus in mammalian development.Mammalian developmental biology is challenged by the need to identify specific developmental regulators. Criteria for identifying the genes that encode these regulators might include the following: (i) involvement in the regulative behavior of mammalian embryos (1), (it) pleiotropic gene action (2), (iii) developmental abnormalities caused by both loss-of-function and gain-of-function alleles (3, 4), and (iv) control over the structure of cell-surface macromolecules (5, 6). Loci of this type may lie in the t region, which extends from the brachyury locus (1) through the major histocompatibility complex (H-2) on murine chromosome 17. Genes in this region strongly affect spermiogenesis (7,8) and the immune system (9, 10).The t region has been a rich source of natural genetic variants (haplotypes), which are frequent in natural populations owing to preferential transmission from the male. The inclusion of detrimental or lethal mutations in a t haplotype may be necessary for the haplotype to remain extant in the mouse population (11). Embryological studies of the initial set of t haplotypes indicated that their lethal effects commonly involve transitions in organization (12), but perhaps the embryo is simply sensitive to any defect in cell metabolism at these organizational stages (13).The t region undoubtedly contains genes of great interest for the study of mammalian developmental biology. However, individual t haplotypes are known to contain multiple lesions (14, 15) and differ by 0.6% in DNA sequence from wild type (16). Furthermore, the recombination-suppression property of t haplotypes because of inversion polymorphism (17) locks together this block of chromatin (=24 x 101 base pairs). The natural t variants, thus, do not give ready access to the simple mutat...

Phenylketonuria (PKU) results from a deficiency in phenylalanine hydroxylase, the enzyme catalyzing the conversion of phenylalanine (PHE) to tyrosine. Although this inborn error of metabolism was among the first in humans to be understood biochemically and genetically, little is known of the mechanism(s) involved in the pathology of PKU. We have combined mouse germline mutagenesis with screens for hyperphenylalaninemia to isolate three mutants deficient in phenylalanine hydroxylase (PAH) activity and cross-reactive protein. Two of these have reduced PAH mRNA and display characteristics of untreated human PKU patients. A low PHE diet partially reverses these abnormalities. Our success in using high frequency random germline point mutagenesis to obtain appropriate disease models illustrates how such mutagenesis can complement the emergent power of targeted mutagenesis in the mouse. The mutants now can be used as models in studying both maternal PKU and somatic gene therapy.