Rat strain variations in liver cytosolic DT-diaphorase activity and possible significance of the trait in carcinogenesis by azo dyes

1. Strain variations among female rats in terms of cytosolic DT-diaphorase activity were studied in liver, heart and glandular stomach tissues with or without administration of 3-tert-butyl-4-hydroxyanisole (BHA). 2. BHA induced liver DT-diaphorase activity in all strains examined, and both the basal and induced activities varied according to strain. Among the five strains tested, Brown Norway (BN) and Sprague-Dawley (SD) rats showed relatively high levels of enzyme activity in the liver, whereas Fischer (F344) rats showed a relatively low level of activity. Results of examination of Fischer-BN-F1 rats indicated that a lower level of liver DT-diaphorase activity was inherited essentially as a dominant trait. 3. Liver DT-diaphorase activity in male rats was significantly lower than in female rats. Small strain variations of the activity, if any, were observed in the heart and stomach cytosolic fractions with or without induction by BHA. The magnitude of induction by BHA was also small, if any, in heart and stomach cytosolic fractions. 4. From these and other observations, we discussed the differences between rats and mice in these strain and tissue variations of DT-diaphorase activity, and also the possible significance of liver DT-diaphorase activity in carcinogenesis by azo dyes.     

Genetic regulation of UDP-glucuronosyltransferase induction by polycyclic aromatic compounds in mice. Co-segregation with aryl hydrocarbon (benzo(alpha)pyrene) hydroxylase induction.

Induction of hepatic 4-methylumbelliferone UDP-glucuronosyltransferase (EC 2.4.1.17) by polycyclic aromatic compounds, such as 3-methylcholanthrene or beta-naphthoflavone, occurs in C57BL/6N, A/J, PL/J, C3HeB/FeJ, and BALB/cJ but not in DBA/2N, AU/SsJ, AKR/J, or RF/J inbred strains of mice. This pattern of five responsive and five nonresponsive mouse strains parallels that of the Ah locus, which controls the induction of aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase (EC 1.14.14.2). Induction of the transferase is maximal in C57BL/6N mice with 200 mg of 3-methylcholanthrene/kg body weight; no induction occurs in nonresponsive DBA/2N mice even at a dose of 400 mg/kg. The rise of inducible transferase activity lags 1 or more days behind the rise of inducible hydroxylase activity and peaks 5 days after a single dose of 3-methylcholanthrene. In offspring from the appropriate backcrosses and intercross between C57BL/6N and DBA/2N parent strains, the genetic expression of 3-methylcholanthrene-inducible transferase activity is inherited as an additive (co-dominant) trait. This expression differs distinctly from that of the inducible hydroxylase activity, which is inherited almost exclusively as a single autosomal dominant trait in these same animals. The more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin induces the transferase more than 3-fold in C57BL/6N mice and less than 2-fold in DBA/2N mice, whereas the hydroxylase is induced equally (about 8-fold) in both strains. A dose of 3-methylcholanthrene given 3 days after 2,3,7,8-tetrachlorodibenzo-p-dioxin, at a time when hydroxylase induction in both strains is very high, does not enhance the rise in inducible transferase activity seen in C57BL/6N or DBA/2N mice which have received 2,3,7,8-tetrachlorodibenzo-p-dioxin alone. These data indicate that (a) the inducibility of two metabolically coordinated membrane-bound enzyme activities may be regulated by a single genetic locus, and (b) although the hydroxylase can be fully induced in the nonresponsive DBA/2N strain by 2,3,7,8-tetrachlorodibenzo-p-dioxin prior to 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene, presumably present in the liver, are incapable of inducing further the transferase activity. The difference in sensitivity between 3-methylcholanthrene and the more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin for both the hydroxylase and the transferase activities suggests the possibility of a common receptor in regulating both enzyme induction processes.     

Induction of multiple chemokine and colony-stimulating factor genes in experimental Burkholderia pseudomallei infection

Melioidosis is a disease of the tropics caused by the facultative intracellular bacterium Burkholderia pseudomallei. In human infection, increased levels of IFN-gamma in addition to the chemokines interferon-gamma-inducible protein 10 (IP-10) and monocyte interferon-gamma-inducible protein (Mig) have been demonstrated. However, the role of these and other chemokines in the pathogenesis of melioidosis remains unknown. Using BALB/c and C57BL/6 mice as models of the acute and chronic forms of human melioidosis, the induction of mRNA was assessed for various chemokines and CSF (G-CSF, M-CSF, GM-CSF, IP-10, Mig, RANTES, MCP-1, KC and MIP-2) in spleen and liver following B. pseudomallei infection. Patterns of chemokine and CSF induction were similar in liver and spleen; however, responses were typically greater in spleen, which reflected higher tissue bacterial loads. In BALB/c mice, high-level expression of mRNA for all chemokines and CSF investigated was demonstrated at day 3 postinfection, correlating with peak bacterial load and extensive infiltration of leucocytes. In contrast, increased mRNA expression and bacterial numbers in C57BL/6 mice were greatest between 4 and 14 days following infection. This paralleled increases in the size and number of abscesses in liver and spleen of C57BL/6 mice at days 3 and 14 postinfection. Earlier induction of cytokine-induced neutrophil chemoattractant (KC), macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) mRNA was demonstrated in spleen, while MIP-2, MCP-1, IP-10 and Mig were demonstrated in liver of BALB/c mice when compared to spleen and liver of C57BL/6. The magnitude of cellular responses observed in the tissue correlated with increased levels of the chemokines and CSF investigated, as well as bacterial load. Compared with C57BL/6 mice, greater infiltration of neutrophils was observed in liver and spleen of BALB/c mice at day 3. In contrast, early lesions in C57BL/6 mice predominantly comprised macrophages. These results suggest that the inability of BALB/c mice to contain the infection at sites of inflammation may underlie the susceptible phenotype of this mouse strain towards B. pseudomallei infection.     

Genetic control of immune responses to the 18-kDa protein of Mycobacterium leprae. Different TH1 subsets may be involved in proliferative and delayed-type hypersensitivity responses.

In vitro and in vivo responses to the 18-kDa protein of Mycobacterium leprae have been analysed in different strains of mice. Lymphocytes from BALB/cJ (H-2d), BALB.B (H-2b), B10.BR (H-2k), and B10.M (H-2f) mice primed with 18-kDa protein yielded high T cell proliferative responses, while those from C57BL/10J (H-2b) mice yielded lower responses. Both H-2 and non-H-2 genes contributed to the magnitude of responsiveness. F1 mice from high and low responder strains showed high responsiveness to the 18-kDa protein. Supernatants from lymph node cell cultures prepared from 18-kDa protein-immunised BALB/cJ, B10.BR, and C57BL/10J mice contained IL-2 but no IL-4, indicating that activated T cells from both high and low responder mice were of a TH1 phenotype. Cell cultures from low responder C57BL/10J mice produced less IL-2 than those from high responders. The low responsiveness to the 18-kDa protein in proliferative assays might be due to a low frequency of antigen-specific T cells in the C57BL/10J mouse strain. BALB/cJ, C57BL/10J, and F1 (BALB/cJ x B10.BR) mouse strains were tested for in vivo DTH reactions to the 18-kDa protein. All strains, including C57BL/10J, were high DTH responders. Although DTH effector cells and 18-kDa protein-specific proliferative T cells belong to the TH1 subset, our data comparing high and low responder status indicate that distinct TH1 subpopulations are stimulated in response to the 18-kDa protein of M. leprae.     

Strain difference in mouse natural killer activity augmented by mouse interferon and poly I.C

The level of natural killer (NK) activity was found to vary considerably among several mouse strains. In vivo and in vitro, interferon (IFN) and IFN inducers have been shown to augment mouse NK activity. C3H/He mice showed high NK activity, DDD/1 and A/J mice low NK activity, and C57BL/6, BALB/c and DBA/2 mice intermediate NK activity after injection with polyinosinic polycytidylic acid (poly I. C.). The same NK activity correlation was observed in nontreated mice, but the NK activities were lower compared with the poly I. C.-injected mice. Moreover, the DDD/1 and A/J mice showed almost no augmentation of NK activity on injection with poly I.C. In vivo, C3H/He, BALB/c and C57BL/6 mice injected with IFN showed augmented NK activity, but DDD/1 mice showed no such reaction. In vitro, C3H/He, BALB/c and C57BL/6 mouse spleen cells treated with IFN also showed augmented NK activity, but DDD/1 mouse spleen cells showed almost none. F1 hybrids between high (C3H/He) and low (DDD/1) NK-activity strains showed high NK activity. Thus, activity is dominant over low activity. The segregation of (DDD/1 X C3H/He) Fl X DDD/1 back-cross mice suggested that the strain differences in NK activity are under polygenic control.     

Genetic control of heat sensitivity and activity level of murine arylsulfatase B

BALB/c male mice possess twofold higher kidney p-nitrocatechol-SO4 arylsulfatase B than do A/HeJ male mice; however, their liver arylsulfatase activities are comparable. Twentyfold-purified kidney arylsulfatases B from these two strains have similar Michaelis constants, electrophoretic mobilities, pH optima, and inhibitor profiles; however, the BALB/c enzyme is more heat stable than the A/HeJ enzyme. BALB/c, C3H/HeJ, DBA/2J, and SWR/J mice share an autosomal allele, As-1a, which apparently determines the heat-stable arylsulfatase B, while A/HeJ and C57BL/6J mice possess the As-1b allele, which determines the heat-sensitive enzyme. A second autosomal locus, Asr-1, determines liver arylsulfatase B activity. C57BL/6J mice carry the Asr-1a allele, which results in high liver activities, while C3H/HeJ mice are homozygous for the low-activity allele, Asr-1b. Male mice generally have 30-40% higher kidney activities than females; however, female kidney arylsulfatase activities rise and actually surpass those of males during late pregnancy and lactation.     

Expression of aryl hydrocarbon hydroxylase induction in mouse tissues in vivo and in organ culture

The elevation of aryl hydrocarbon hydroxylase by various microsomal enzyme inducers in mouse tissues from five inbred strains was examined in vivo and in fetal liver expiants. The magnitude of 3-methylcholanthrene- or β-naphthoflavone-inducible AHH activities in the intact animal varied greatly with the tissue and strain—from no induction in the liver and less than a 2- to 3-fold increase in the lung of DBA/2+ and AKR mice to 4- to 5- and 6- to 7-fold elevation, respectively, in the liver and lung of C57BL mice. Treatment of At or C3H⁺ mice with these inducers increased AHH activity in liver and lung to levels which were intermediate between those observed with tissues from DBA/2+ and C57BL mice. These strain-specific differences in the expression of AHH induction in response to polycyclic hydrocarbons and flavones were also present in fetal liver expiants and were measurable as early as 6 days before parturition. In expiants derived from polycyclic hydrocarbon-“responsive” strains, the extent of enzyme induction was greatest with 4′-bromoflavone, less with β-naphthoflavone and least with 3-methylcholanthrene. Trans-1, 2-dihydroxy-3-methylcholanthrene was about twice as effective in this regard as the parent compound 3-methylcholanthrene. Among expiants from 3-methylcholanthrene-“resistant” strains (DBA/2+, AKR), a disparity in the effects of different classes of compounds was apparent: the flavone derivatives induced aryl hydrocarbon hydroxylase activity from DBA/2+ and AKR expiants by 2- to 3-fold despite the absence of polycyclic hydrocarbon induction in these cultures. Furthermore, although phenobarbital was a comparatively weak inducer under the conditions used in these experiments, this substance stimulated aryl hydrocarbon hydroxylase activity from 3-methylcholanthrene-“responsive” and -“resistant” explants by similar degrees (i.e., about 30%). The results are discussed in the light of previous suggestions on the genetically determined regulation of aryl hydrocarbon hydroxylase induction in mouse tissues.     

Reduction of Brain Antioxidant Defense Upon Treatment with Butylated Hydroxyanisole (BHA) and Sudan III in Syrian Golden Hamster

Treatment with the antioxidant butylated hydroxyanisole (BHA) or the azo dye Sudan III during two weeks led to changes in the brain enzymatic antioxidant defense of Syrian golden hamsters. BHA was able to induce liver superoxide dismutase (SOD) 2-fold but had no effect on the brain SOD activity, whereas SOD activity was reduced to 50% in brain and remained unchanged in liver with Sudan III. These two substances are known inducers of DT-diaphorase and in fact this enzymatic activity was induced 4- and 6-fold in liver with BHA and Sudan III, respectively. However, BHA promoted a significant 40% reduction, whereas no change was observed with Sudan III in brain DT-diaphorase activity. Glutathione(GSH)-related enzymatic activities were also assayed in brain and liver. No induction was observed with BHA or Sudan III for any of the activities tested in hamster brain: GSH S-transferase (GST), GSH peroxidase (GSH-Px) and glutathione disulfide (GSSG) reductase (GR). Only 1.3- and 1. 4-fold increases of GST and GR activities were observed in liver and no change in any of these enzymatic activities in brain with BHA; a partial limitation of permeability to BHA of the blood-brain barrier may explain this results. Furthermore, Sudan III promoted reductions in all these GSH-related enzymatic activities in brain and liver. The possible explanations for these results are discussed.Deceased 4th November 1998     

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.     

Cytokines regulate the pattern of rejection and susceptibility to cyclosporine therapy in different mouse recipient strains after cardiac allografting

We determined the role of cytokines in regulating the pattern of rejection and recipient susceptibility to cyclosporine (CsA) in a mouse cardiac allograft model. Hearts from C3H mice transplanted into untreated BALB/c (Th2-dominant) and C57BL/6 (Th1-dominant) mice showed different patterns of rejection. C3H allografts in BALB/c mice showed typical acute vascular rejection (AVR) with strong intragraft deposition and high serum levels of anti-donor IgG with predominant IgG1, while C3H allografts in C57BL/6 mice showed typical acute cellular rejection (ACR) with massive intragraft infiltration of CD4(+) and CD8(+) lymphocytes and low serum levels of anti-donor IgG with predominant IgG2a. Elevated intragraft mRNA expression of IL-2, IFN-gamma, and IL-12 mRNA was present in C57BL/6 recipients, whereas allografts in BALB/c mice displayed increased IL-4 and IL-10 mRNA levels. CsA therapy completely inhibited ACR and induced indefinite allograft survival in C57BL/6 recipients, while the same therapy failed to prevent AVR, and only marginally prolonged graft survival in BALB/c recipients. In contrast, rapamycin blocked AVR, achieving indefinite survival in BALB/c recipients, but was less effective at preventing ACR in C57BL/6 recipients. The disruption of the IL-12 or IFN-gamma genes in C57BL/6 mice shifted ACR to AVR, and resulted in concomitant recipient resistance to CsA therapy. Conversely, disruption of IL-4 gene in BALB/c mice markedly attenuated AVR and significantly prolonged allograft survival. These data suggest that the distinct cytokine profiles expressed by different mouse strains play an essential role in regulating the pattern of rejection and outcome of CsA/rapamycin therapy.     

Taste synergism between monosodium glutamate and 5'-ribonucleotide in mice.

1. Strain differences of mice were found in the taste synergism between monosodium L-glutamate (MSG) and disodium 5'-guanylate (GMP). 2. Magnitudes of chorda tympani responses to the mixture of MSG and GMP over the sum of responses to each component were greater in the order of C3H/HeSlc(C3H) greater than C57BL/6CrSlc(C57BL) greater than BALB/cCrSlc(BALB) mice. The greatest synergism was observed in response to the mixture of 0.03 M MSG and 0.1 mM GMP, to which responses were about 2.6, 1.8 and 1.4 times greater than the sum of each component in C3H, C57BL and BALB mice, respectively. 3. Magnitudes of inhibition of MSG and mixture responses by the lingual treatment of proteolytic enzyme, Pronase E, were greater in the same order of C3H greater than C57BL greater than BALB mice as that observed in magnitudes of the synergism. These results suggest that there exists quantitative differences in receptors responsible for taste synergism between MSG and GMP among three mouse strains.     

Age and strain dependence of O6-methylguanine DNA methyltransferase activity in mice

The activity of the DNA repair protein O6-methylguanine DNA methyltransferase (MT) was compared in liver extracts from female ICR and male C57BL/6 mice at various ages (3-130 weeks old). Similar patterns of overall enzyme activity were observed in both strains with O6-MT activity being relatively low in young mice (3 or 8 weeks old). However, the activity significantly increased after adolescence (middle age), thereafter decreasing with old age (over 100 weeks old) to a level equivalent to that found in young mice. In an additional strain difference study, O6-MT activities in liver extracts from 4 strains of mice were compared at 5 and 30 weeks of age. Although a similar age-associated increase of enzyme activity in adolescence was confirmed in all 4 strains investigated, the closed-colony ICR mice differed from the inbred strains in demonstrating significantly higher levels of O6-MT activity in females than in males. However, the same tendency was also observed in a comparison of the sexes in 30-week-old C3H/HeN, C57BL/6 and BALB/c mice.     

Studies on pulmonary aryl hydrocarbon hydroxylase activity in inbred strains of mice.

Pulmonary and hepatic levels of aryl hydrocarbon hydroxylase (AHH) were studied in inbred strains of mice following intratracheal (i.t.) instillation of 3-methylcholanthrene (MCA). I.t. instillation of 188 mug MCA in sterile 0.2% gelatin in saline resulted in preferential induction of pulmonary AHH. After treatment with this dose of MCA, the pulmonary AHH levels of strains C57BL/6Cum, C57BL/6J, BALB/cMai, C3H/fMai, and C57L/J were observed to be induced within 24 h after treatment. Strains DBA/2Cum, AKR/J, SJL/J, DBA/2J and RF/J expressed no such increase. At a dose of 500 mug MCA, the pulmonary tissue of DBA/2 mice did express a 4-fold increase. This increase in AHH was determined to be quite different from the increase observed in C57BL/6 mice by: (1) specific activity of the enzymes, (2) genetic regulation, (3) susceptibility to inhibition by 7,8-benzoflavone, and (4) spectral properties of the associated cytochromes. It was of major importance that induction of pulmonary AHH was observed to be regulated by a single dominant gene in crosses involving the C57BL/6Cum and DBA/2Cum strains of mice. Results were discussed with the view in mind that these genetically regulated levels of AHH may play a role in susceptibility to cancers induced by polycyclic aromatic hydrocarbon carcinogens.     

Isosafrole-induced cytochrome P2-450 in DBA/2N mouse liver. Characterization and genetic control of induction

Mouse "cytochrome P2-450" is defined as that form of isosafrole-induced P-450 in DBA/2N liver most specifically correlated with isosafrole metabolism. Isosafrole pretreatment does not induce aryl hydrocarbon hydroxylase activity ("cytochrome P1-450") in C57BL/6N or DBA/2N mice, induces acetanilide 4-hydroxylase activity ("cytochrome P3-450") more than 3-fold in C57BL/6N but not in DBA/2N mice, and induces isosafrole metabolite formation more than 3-fold in both C57BL/6N and DBA/2N mice. P2-450 was, therefore, purified from isosafrole-treated DBA/2N liver microsomes having negligible amounts of contaminating P1-450 and P3-450. The apparent molecular weight of P2-450 is 55,000, and the protein appears homogeneous on sodium dodecyl sulfate-polyacrylamide gels. The Soret peak of the reduced purified cytochrome X CO complex is 448 nm. Purified P2-450, reconstituted in vitro, metabolizes acetanilide poorly and benzo[a]pyrene hardly at all. Anti-(P2-450) inhibits (90 to 100%) liver microsomal isosafrole metabolite formation, yet has no effect on aryl hydrocarbon hydroxylase, acetanilide 4-hydroxylase, biphenyl 2- or 4-hydroxylase, or 7-ethoxycoumarin O-de-ethylase activities. 3-Methylcholanthrene induces anti-(P2-450)-precipitable protein about 12-fold in C57BL/6N and 2-fold in DBA/2N liver; 2,3,7,8-tetrachlorodibenzo-p-dioxin (10 micrograms/kg), about 12-fold in both C57BL/6N and DBA/2N liver; isosafrole, more than 3-fold in both C57BL/6N and DBA/2N. Benzo[a]anthracene at maximal doses induces anti-(P2-450)-precipitable protein in C57BL/6N liver no more than 2-fold, yet is known to be a highly potent inducer of P1-450 mRNA in C57BL/6N liver. The sensitivity of the P2-450 induction process to isosafrole is inherited as an autosomal additive trait; studies of offspring from the C57BL/6N(DBA/N)F1 X DBA/2N backcross confirm involvement of the Ah locus or s closely segregating gene. In contrast, among crosses between C57BL/6N and DBA/2N, sensitivity of the P1-450 and P3-450 induction process to 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin is inherited as an autosomal dominant trait. These data suggest that, although P1-450, P2-450, and P3-450 proteins are controlled by the Ah locus, either a P-450 protein polymorphism exists between C57BL/6N and DBA/2N mice or subtle differences may exist in the interaction of various inducers with Ah receptor.     

Genetic control of glycolipid expression

A polymorphic variation of sialic acid species of sialosyllactosylceramide was found in dog erythrocytes. The analysis of the glycolipids in the erythrocytes of the individual dogs in a family of a Japanese breed of dog, Shiba-Inu, showed that the expression of sialosyllactosylceramide containing N-glycolylneuraminic acid was an autosomal dominant trait over the expression of that containing N-acetylneuraminic acid. Polymorphic variations of major liver gangliosides were also found in various strains of inbred mice. The strains were classified into three groups; the first group possessed only II3 NeuGc-LacCer, the second group possessed II3NeuGc-GgOse3Cer in addition to II3NeuGc-LacCer and the third group possessed II3NeuGc-GgOse4Cer and II3NeuGc,IV3NeuGc-GgOse4Cer as well as the above two gangliosides. By subjecting mice of these three groups to genetic analysis, the strain of the first group (WHT/Ht mice) was demonstrated to be a recessive homozygote which had a single autosomal defective gene making it unable to express N-acetylgalactosaminyltransferase activity to produce II3NeuGc-GgOse3Cer. The strains of the second group (BALB/c and C57BL/10 mice) were also demonstrated to be recessive homozygotes which had a single autosomal defective gene making them unable to express high enough level of galactosyltransferase activity to produce II3NeuGc-GgOse4Cer. By the analysis of gangliosides and the enzyme activity of H-2 congenic mice and mice produced by a mating, this defective gene controlling the expression of II3NeuGc-GgOse4-Cer through the regulation of the transferase activity was demonstrated to be linked to H-2 complex on chromosome 17.     

Confirmation and high resolution mapping of an atherosclerosis susceptibility gene in mice on Chromosome 1

Previously, we demonstrated that Ath1 is a quantitative trait locus for aortic fatty streak formation, located on Chromosome (chr) 1, with susceptibility in C57BL/6J mice and resistance in C3H/HeJ and BALB/cJ mice fed an atherogenic diet. In this study, we find an atherosclerosis susceptibility locus in the same region of Chr 1 by constructing two congenic strains with the resistance phenotype transferred from different resistant strains, PERA/EiJ or SPRETUS/EiJ. By backcrossing one congenic strain to C57BL/6J and testing recombinant animals, we reduced the distance of the atherosclerosis susceptibility region to 2.3 cM between D1Mit14 and D1Mit10. Further testing of nine recombinant animals showed that eight of the nine were consistent with a further narrowing between D1Mit159 and D1Mit398 a distance of 0.66 cM. This region encompasses a number of potential candidate genes including the thiol-specific antioxidant gene Aop2, also known as peroxiredoxin 5 (Prdx5). AOP2 is capable of reducing hydroperoxides and lipid peroxides in the cell. To investigate Aop2 as a potential candidate, we mapped Aop2 in our backcross and localized it to the atherosclerosis susceptibility interval. We determined that Aop2 is highly expressed in atherosclerosis-related tissues including liver and heart. We also found an inverse correlation between Aop2 mRNA in liver and atherosclerosis phenotype for strains C57BL/6 and the resistant congenic derived from SPRETUS/EiJ. Since LDL oxidation has been implicated in the pathogenesis of this disease, and AOP2 possesses antioxidant activity, we suggest the role of Aop2 in atherosclerosis susceptibility needs to be further explored.     

Gene expression profiles in genetically different mice infected with Toxoplasma gondii: ALDH1A2, BEX2, EGR2, CCL3 and PLAU

Toxoplasma gondii can modulate host cell gene expression; however, determining gene expression levels in intermediate hosts after T. gondii infection is not known much. We selected 5 genes (ALDH1A2, BEX2, CCL3, EGR2 and PLAU) and compared the mRNA expression levels in the spleen, liver, lung and small intestine of genetically different mice infected with T. gondii. ALDH1A2 mRNA expressions of both mouse strains were markedly increased at day 1-4 postinfection (PI) and then decreased, and its expressions in the spleen and lung were significantly higher in C57BL/6 mice than those of BALB/c mice. BEX2 and CCR3 mRNA expressions of both mouse strains were significantly increased from day 7 PI and peaked at day 15-30 PI (P<0.05), especially high in the spleen liver or small intestine of C57BL/6 mice. EGR2 and PLAU mRNA expressions of both mouse strains were significantly increased after infection, especially high in the spleen and liver. However, their expression patterns were varied depending on the tissue and mouse strain. Taken together, T. gondii-susceptible C57BL/6 mice expressed higher levels of these 5 genes than did T. gondii-resistant BALB/c mice, particularly in the spleen and liver. And ALDH1A2 and PLAU expressions were increased acutely, whereas BEX2, CCL3 and EGR2 expressions were increased lately. Thus, these demonstrate that host genetic factors exert a strong impact on the expression of these 5 genes and their expression patterns were varied depending on the gene or tissue.     

Efficiency alleles of the Pctr1 modifier locus for plasmacytoma susceptibility

The susceptibility of BALB/c mice to pristane-induced plasmacytomas is a complex genetic trait involving multiple loci, while DBA/2 and C57BL/6 strains are genetically resistant to the plasmacytomagenic effects of pristane. In this model system for human B-cell neoplasia, one of the BALB/c susceptibility and modifier loci, Pctr1, was mapped to a 5.7-centimorgan (cM) chromosomal region that included Cdkn2a, which encodes p16(INK4a) and p19(ARF), and the coding sequences for the BALB/c p16(INK4a) and p19(ARF) alleles were found to be polymorphic with respect to their resistant Pctr1 counterparts in DBA/2 and C57BL/6 mice (45). In the present study, alleles of Pctr1, Cdkn2a, and D4Mit15 from a resistant strain (BALB/cDAG) carrying DBA/2 chromatin were introgressively backcrossed to the susceptible BALB/c strain. The resultant C.DAG-Pctr1 Cdkn2a D4Mit15 congenic was more resistant to plasmacytomagenesis than BALB/c, thus narrowing Pctr1 to a 1.5-cM interval. Concomitantly, resistant C57BL/6 mice, from which both gene products of the Cdkn2a gene have been eliminated, developed pristane-induced plasma cell tumors over a shorter latency period than the traditionally susceptible BALB/cAn strain. Biological assays of the p16(INK4a) and p19(ARF) alleles from BALB/c and DBA/2 indicated that the BALB/c p16(INK4a) allele was less active than its DBA/2 counterpart in inducing growth arrest of mouse plasmacytoma cell lines and preventing ras-induced transformation of NIH 3T3 cells, while the two p19(ARF) alleles displayed similar potencies in both assays. We propose that the BALB/c susceptibility/modifier locus, Pctr1, is an "efficiency" allele of the p16(INK4a) gene.     

Tyrosine hydroxylase in various brain regions of three strains of mice differing in spontaneous activity, learning ability, and emotionality.

Tyrosine hydroxylase has been measured in brains of three inbred strains of mice ; DBA/2J ; C57 BL/6J and BALB/cJ. Compared to C57 BL/6J, DBA/2J showed a higher enzyme activity in hypothalamus, a lower activity in pons-medulla, and no significant changes in cortex or striatum. BALB/cJ showed a higher level of activity in all regions studied (striatum, pons-medulla and hypothalamus). No effect of isolation or of social dominance position were noted on the enzyme activities in C57 BL/6J or BALB/cJ mice.     

Participation of embryonic genotype in the pregnancy block phenomenon in mice.

Pregnancy block by male pheromones in mice differs in incidence depending on the combination of strains. Female mice of BALB/cA strain mated with BALB/cA males show a 100% pregnancy block when exposed to males of inbred strain DDK shortly after copulation (Chung et al., Biol Reprod 1997; 57:312-319). In the present study, BALB/cA females mated with the males of other strains--CBA/J, C3H/HeN, C57BL/6Cr, and IXBL--showed higher pregnancy rates (66.6-87. 5%) even when they were exposed to DDK males. In the pharmacological induction of pregnancy block with dopamine agonist (bromocriptine, 4 mg/kg BW), BALB/cA females mated with BALB/cA males showed a 100% pregnancy block. In contrast, BALB/cA females mated with CBA/J, C3H/HeN, and C57BL/6Cr males showed higher pregnancy rates (40-70%). These results suggest that the better pregnancy rate of BALB/cA females mated with alien males may be due to the stronger viability of F(1) embryos. This interpretation was confirmed by an embryo transfer experiment in which a higher implantation rate was observed when BALB/cA embryos grown in BALB/cA females exposed to BALB/cA males were transferred into recipient BALB/cA females exposed to DDK males. These results suggest that the embryonic genotype or viability of the embryo is one factor contributing to the occurrence of pregnancy block by male pheromones in mice.