C3H/HeJ Strains Research Articles

Generalized resistance to pruritogen-induced scratching in the C3H/HeJ strain.

Previously the effect of the pruritogens, such as histamine and chloroquine, was tested in 11 inbred mouse strains, and this study aimed to identify resistant and sensitive strains, consistent with the observation that underlies the large variability in human populations. In the present study, we used the low responder C3H/HeJ (C3H) and the more sensitive C57BL/6J (C57) strain to find out if resistance and sensitivity to develop pruritus is restricted to only histamine and chloroquine or extends to other known pruritogens as well. We tested five additional commonly known pruritogens. We established dose-response relationships by injecting four concentrations of the pruritogens in the range of 0.3, 1, 3, and ten-fold in the nuchal fold. Then we assessed the scratching behavior for 30 min after injection with an automated custom-designed device based on the bilateral implantation of mini-magnets in the hind paws and on single cages placed within a magnetic coil. We found that the resistance to pruritogens is a general phenotype of the C3H strain and extends to all pruritogens tested, including not only histamine and chloroquine, but also endothelin, trypsin, 5-HT (serotonin), the short peptide SLIGRL, and Lysophosphatidic acid (LPA). C57 was more sensitive to all pruritogens and, in contrast to C3H, dose-response relationships were evident for some of the pruritogens. In general, comparable peak scratch responses were observed for the 0.3-fold concentrations of the pruritogens in C57 whereas C3H required at least the ten-fold concentration and still displayed only between 5 and 33% of the scratch responses observed in C57 for the respective pruritogen. The general resistance to pruritogens and the low level of scratching behavior found in the C3H strain is an interesting trait and represents a model for the study of the heritability of itch. It is accompanied in C3H with a higher sensitivity in assays of nociception.

Mechanical loading of mouse caudal vertebrae increases trabecular and cortical bone mass-dependence on dose and genotype

Most in vivo studies addressing the skeletal responses of mice to mechanical loading have targeted cortical bone. To investigate trabecular bone responses also we have developed a caudal vertebral axial compression device (CVAD) that transmits mechanical loads to compress the fifth caudal vertebra via stainless steel pins inserted into the forth and sixth caudal vertebral bodies. Here, we used the CVAD in C57BL/6 (B6) and C3H/Hej (C3H) female mice (15weeks of age) to investigate whether the effect of regular bouts of mechanical stimulation on bone modeling and bone mass was dependent on dose and genotype. A combined micro-computed tomographic and dynamic histomorphometric analysis was carried out at the end of a 4-week loading regimen (3,000 cycles, 10 Hz, 3 xweek) for load amplitudes of 0N, 2N, 4N and 8N. Significant increases in trabecular bone mass of 9 and 21% for loads of 4N and 8N, respectively, were observed in B6 mice. A significant increase of 10% in trabecular bone mass occurred for a load of 8N in the C3H strain. For other loads, no significant increases were detected. Both mouse strains exhibited substantial increases in trabecular bone formation rates for all loads, B6: 111% (2N), 86% (4N), 164% (8N), C3H: 41% (2N), 38% (4N), 141% (8N). Significant decreases in osteoclast number of 146 and 93% for a load of 8N were detected in B6 and C3H mice, respectively. These findings demonstrate that the effect of loading on the structural and functional parameters of bone is dose and genotype dependent. The caudal vertebral loading model established here is proposed for further studies addressing the molecular processes involved in the skeletal responses to mechanical stimuli.

Modulation of murine bone marrow‐derived CFU‐F and CFU‐OB by in vivo bisphosphonate and fluoride treatments

Bisphosphonates (BPN) have actions on a variety of cell types including: osteoclasts, osteoblasts, osteocytes, and endothelial cells. The objectives of this report are to review the current state of understanding of the effects of BPNs on orthodontic tooth movement and to provide evidence on BPN's in vivo effects on bone marrow-derived osteoprogenitor cells. Mice from the C3H/HeJ (C3H), C57BL/6J (B6), FVB/NJ (FVB), and BALB/cByJ (BALB) strains were treated for 3 weeks with 0, 3, 30, or 150 mcg/kg/week alendronate (ALN) administered subcutaneous alone or in combination with 50 ppm fluoride (F). Bone marrow cells were harvested and subjected to in vitro colony-forming unit fibroblast (CFU-F) and colony-forming unit osteoblasts (CFU-OB) assays. Baseline differences in CFU-F, CFU-OB/ALP+, and CFU-OB/total were observed among the four strains. Strain-specific responses to ALN and F treatments were observed for CFU-F, CFU-OB/ALP+, and CFU-OB/total. F treatment alone resulted in decreases in CFU-F (p = 0.013), CFU-OB/ALP+ (p = 0.005), and CFU-OB/total (p = 0.003) in the C3H strain. CFU-F (p = 0.036) were decreased by F in the B6 strain. No significant (NS) effects of F were observed for FVB and BALB. ALN treatment resulted in a significant decrease in CFU-F (p = 0.0014) and CFU-OB/total (p = 0.028) in C3H only. ALN treatment had NS effect on CFU-OB/ALP+ in all four strains. Genetic factors appear to play a role in ALN's effects on CFU-F and CFU-OB/total but not on CFU-OB/ALP+.

Variable Effects of Caloric Restriction on Metabolic and Breathing Regulation Between Two Inbred Mouse Strains

The World Health Organization reports that ~800 million people worldwide are undernourished, corresponding to a daily mortality rate of 25,000 people. While altered metabolic and breathing characteristics occur with severe caloric restriction (CR), the mechanisms are unclear. We hypothesize that a robust genetic difference between C3H/HeJ (C3) and C57BL/6 (B6) mice lead to variation in metabolic and breathing regulation with CR. C3 and B6 mice (n = 8 per strain) were fed a diet equal to 33% of the daily food intake for 21 days. Using plethysmography, breathing characteristics were measured weekly on room air, and following acute hypercapnic (5% CO2) and hypoxic (10% O2) challenges. While the decline in body weight through 21 days of CR was similar in C3 vs. B6 strains (~40%), reductions in body temperature (10.0±1.8 vs. 2.8±1.0°C) and O2 consumption (1.36±0.32 vs. ‐0.04±0.16 ml/hr/g) were significantly (P < 0.01) lower in C3 relative to B6 mice. Minute ventilation (VE) on room air remained unchanged with CR in both strains, but hypercapnic and hypoxic VE were depressed incrementally though 14 days. By day 21 of CR, hypercapnic and hypoxic VE recovered in B6 but not in C3 mice. In conclusion, genetic factors determine variation in metabolic and breathing regulation during CR, allowing B6 mice to maintain indices of homeostasis closer to control values than C3 mice.

Genetic Variation in the Patterns of Skeletal Progenitor Cell Differentiation and Progression During Endochondral Bone Formation Affects the Rate of Fracture Healing

These studies examined how genetic differences that regulate architectural and bone material properties would be expressed during fracture healing and determine whether any of these features would affect rates of healing as defined by regain of strength. Controlled fractures were generated in three inbred strains of mice: A/J, C57Bl/6J (B6), and C3H/HeJ (C3H). Both the A/J and B6 strains showed faster healing than the C3H strain based on regains in strength and stiffness. Strain-specific architectural features such as moment of inertia, cross-sectional area, and cortical thickness were all recapitulated during the development of the callus tissues. None of these traits were directly relatable to rates of fracture healing. However, rates of healing were related to variations in the temporal patterns of chondrogenic and osteogenic lineage development. The B6 strain expressed the highest percentage of cartilage gene products and had the longest period of chondrocyte maturation and hypertrophy. The slowest healing strain (C3H) had the shortest period of chondrogenic development and earliest initiation of osteogenic development. Although the A/J strain showed an almost identical pattern of chondrogenic development as the C3H strain, A/J initiated osteogenic development several days later than C3H during fracture healing. Long bone growth plates at 28 days after birth showed similar strain-specific variation in cartilage tissue development as seen in fracture healing. Thus, the B6 strain had the largest growth plate heights, cell numbers per column, and the largest cell size, whereas the C3H columns were the shortest, had the smallest number of cells per column, and showed the smallest cell sizes. These results show that (1) different strains of mice express variations of skeletal stem cell lineage differentiation and (2) that these variations affect the rate of fracture healing.

Candidate genes controlling pulmonary function in mice: transcript profiling and predicted protein structure

Impaired development and reduced lung capacity are risk factors of asthma and chronic obstructive pulmonary disease. Previously, our genomewide linkage analysis of C3H/HeJ (C3H) and JF1/Msf (JF1) mouse strains identified quantitative trait loci (QTLs) associated with the complex traits of dead space volume (Vd), total lung capacity (TLC), lung compliance (CL), and diffusing capacity for CO (D(CO)). We assessed positional candidate genes by comparing C3H with JF1 lung transcript levels by microarray and by comparing C3H, BALB/cByJ, C57BL/6J, A/J, PWD/PhJ, and JF1 strains, using exon sequencing to predict protein structure. Microarray identified >900 transcripts differing in C3H and JF1 lungs related to lung development, function, and remodeling. Of these, three genes localized to QTLs associated with differences in lung function. C3H and JF1 strains differed in transcript and protein levels of superoxide dismutase 3, extracellular [SOD3; mouse chromosome (mCh) 5: VD] and transcript of trefoil factor 2 (TFF2; mCh 17: TLC and D(CO)), and ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2; mCh 15: TLC and CL). Nucleotide sequencing of Sod3, Tff2, and previously identified Relaxin 1 (Rln1; mCh 19: CL) uncovered polymorphisms that could lead to nonsynonymous amino acid changes and altered predicted protein structure. Gene-targeted Sod3(-/-) mice had increased conducting airway volume (Vd/TLC) compared with strain-matched control Sod3(+/+) mice, consistent with the QTL on mCh 5. Two novel genes (Tff2 and Enpp2) have been identified and two suspected genes (Sod3 and Rln1) have been supported as determinants of lung function in mice. Findings with gene-targeted mice suggest that SOD3 is a contributing factor defining the complex trait of conducting airway volume.

Genetic background influences fluoride's effects on osteoclastogenesis

Excessive fluoride (F) can lead to abnormal bone biology. Numerous studies have focused on the anabolic action of F yet little is known regarding any action on osteoclastogenesis. Little is known regarding the influence of an individual's genetic background on the responses of bone cells to F. Four-week old C57BL/6J (B6) and C3H/HeJ (C3H) female mice were treated with NaF in the drinking water (0 ppm, 50 ppm and 100 ppm F ion) for 3 weeks. Bone marrow cells were harvested for osteoclastogenesis and hematopoietic colony-forming cell assays. Sera were analyzed for biochemical and bone markers. Femurs, tibiae, and lumbar vertebrae were subjected to microCT analysis. Tibiae and femurs were subjected to histology and biomechanical testing, respectively. The results demonstrated new actions of F on osteoclastogenesis and hematopoietic cell differentiation. Strain-specific responses were observed. The anabolic action of F was favored in B6 mice exhibiting dose-dependent increases in serum ALP activity ( p < 0.001); in proximal tibia trabecular and vertebral BMD (tibia at 50&100 ppm, p = 0.001; vertebrae at 50 and 100 ppm, p = 0.023&0.019, respectively); and decrease in intact PTH and sRANKL ( p = 0.045 and p < 0.001, respectively). F treatment in B6 mice also resulted in increased numbers of CFU-GEMM colonies ( p = 0.025). Strain-specific accumulations in bone [F] were observed. For C3H mice, dose-dependent increases were observed in osteoclast potential ( p < 0.001), in situ trabecular osteoclast number ( p = 0.007), hematopoietic colony forming units (CFU-GEMM: p < 0.001, CFU-GM: p = 0.006, CFU-M: p < 0.001), and serum markers for osteoclastogenesis (intact PTH: p = 0.004, RANKL: p = 0.022, TRAP5b: p < 0.001). A concordant decrease in serum OPG ( p = 0.005) was also observed. Fluoride treatment had no significant effects on bone morphology, BMD, and serum PYD cross-links in C3H suggesting a lack of significant bone resorption. Mechanical properties were also unaltered in C3H. In conclusion, short term F treatment at physiological levels has strain-specific effects in mice. The expected anabolic effects were observed in B6 and novel actions hallmarked by enhanced osteoclastogenesis shifts in hematopoietic cell differentiation in the C3H strain.

Genetic variability of induction and emergence times for inhalational anaesthetics

Anaesthetic requirements differ among inbred mouse strains. We tested the genetic influence on induction and arousal times to inhalational anaesthetics in two of these strains. Five male C57BL/6J (B6) and five male C3H/HeJ (C3) mice were each exposed to five different concentrations of nitrous oxide (N2O) at five different levels of halothane. Time to sleep and arousal were assessed. Data were analysed by repeated measures of analysis of variance. Halothane, N2O and genetic strain, all were significant independent factors on the time to sleep, while only N2O was a significant independent factor on the time to arousal (P = 0.004). B6 mice took significantly longer to fall asleep compared to the C3 mice controlling for halothane and N2O concentrations (F-ratio = 36, P < 0.0001). The effect of N2O on time to arousal was only significant for the B6 strain (F-ratio = 10, P = 0.005), and not for the C3 strain (F-ratio = 0.8, P = 0.38). Genetics influences the time to sleep for anaesthetic agents in mice.

Effect of MAO A deficiency on different kinds of aggression and social investigation in mice

Monoamine oxidase A (MAO A) degrades serotonin, dopamine and noradrenaline, factors critically involved in the regulation of aggression. Different kinds of aggression were investigated in Tg8, a transgenic mouse strain lacking a functional MAO A gene. MAO A-deficient mice differ from wild-type C3H/HeJ (C3H) in terms of showing higher territorial, predatory and isolation-induced aggression. Tg8 demonstrated shorter latencies to cricket killing and to the first attack after 6 weeks isolation than C3H mice. In the resident-intruder paradigm, MAO A-lacking mice were more aggressive than C3H when tested as intruders. In contrast to C3H, attack in Tg8 mice did not depend on different aggressiveness of intruders of BALB/c, A/Sn and C3H strains. Tg8 mice displayed no increase in aggression but demonstrated reduced social investigation towards anesthetized, as well as towards juvenile BALB/c males. Thus, MAO A deficiency in Tg8 mice is accompanied by increased expression of different kinds of aggression, as well as by disruption of normal pattern of social interaction.

Genetic differences in the IGF-I gene among inbred strains of mice with different serum IGF-I levels

There is significant heterogeneity in serum IGF-I concentrations among normal healthy individuals across all ages and among inbred strains of mice. C3H/HeJ (C3H) mice have 30% higher serum IGF-I concentrations over a lifetime than C57BL/6J (B6), even though body size and length are identical. The underlying mechanism for this disparity remains unknown although several possibilities exist including altered GH secretion, resistance to GH action, or impaired IGF-I secretion from the liver or peripheral tissues. To study this further, we evaluated mRNA levels of pituitary GH, and of IGF-I, GH receptor (GHR) and acid-labile subunit (ALS) in liver and skeletal muscle of male C3H and B6 strains. mRNA levels of hepatic IGF-I paralleled serum IGF-I levels, whereas pituitary GH mRNA expression was significantly lower in C3H than B6. In addition, reduced hepatic mRNA levels of ALS and GHR in B6 suggests hepatic GH resistance in B6. In contrast, mRNA levels of IGF-I and GHR in skeletal muscle were not different between B6 and C3H. There was a single sequence repeat polymorphism (SSR) in the promoter region of both GHR and IGF-I genes in mice; the SSR in the IGF-I gene was significantly different between the two strains. The SSR in the IGF-I gene corresponds to the E2F binding site, which is critical for regulating IGF-I gene expression. These results suggest that the SSR in the promoter region of the IGF-I gene may be partially responsible for differences in serum IGF-I levels between B6 and C3H strains.

MAO A knockout attenuates adrenocortical response to various kinds of stress

The effect of a lack of the gene encoding monoamine oxidase A (MAO A) in transgenic Tg 8 mice on the corticosterone response to restraint, cold, water deprivation-induced, or social acute stress as well as chronic variable stress was studied. It was found that Tg 8 mice with genetic MAO A knockout and wild-type C3H/HeJ (C3H) strain showed similar plasma corticosterone resting level. MAO A knockout mice differed from C3H mice by attenuated response to restraint (60 min), cold (4 degrees C, 60 min), and water deprivation (48 h) as well as to a chronic (15 days) variable stress. No difference between Tg 8 and C3H strains in the response to psychosocial stress (encounters for 30 min of six previously isolated mice) has been found. ACTH administration to dexamethasone-pretreated mice produced a similar corticosterone effect in Tg 8 and C3H mice, indicating that the decreased stress response in MAO A-deficient mice was due rather to the central mechanisms regulating stress-induced ACTH release than to adrenocortical responsiveness to ACTH.

Radiation-induced strain differences in mouse alveolar inflammatory cell apoptosis

Whole-thorax irradiation results in the development of the diffuse inflammatory response alveolitis in C3H/HeJ (C3H) mice and a milder alveolitis with fibrosis in C57BL/6J (B6) mice. In this study, we investigate if this mouse strain difference in response to radiation is due to differences in lung inflammatory cell apoptosis. Mice of the C3H and B6 strains were given a radiation dose of 18 Gy to the thorax and the animals were sacrificed at 11 or 18 weeks following exposure or when they were moribund. Active caspase-3 staining was used to identify apoptotic cells in the alveolar space of histological lung sections from the mice. The apoptotic index of B6 mice was greater than that of C3H mice at 11 weeks postirradiation (17.8% of airspace cells vs. 7.8%, p = 0.028) and in mice sacrificed because of illness (27.3% vs. 14.4%, p = 0.036). No C3H mice survived to the later time point. The inflammatory cells undergoing apoptosis in the mouse lungs were morphologically consistent with alveolar macrophages. We conclude that a difference in inflammatory cell apoptosis may contribute to the disparate pulmonary radiation response of these mouse strains.

Analysis of reciprocal congenic lines reveals the C3H/HeJ genome to be highly resistant to ApcMin intestinal tumorigenesis

Abstract Genetic background affects polyp development in the Multiple intestinal neoplasia ( Apc Min ) mouse model. The Modifier of Min 1 ( Mom1) locus accounts for ~50% of the variation in polyp multiplicity. We generated reciprocal congenic lines, such that the recipient C57BL/6J (B6) strain carries a donor C3H/HeJ (C3H) Mom1 allele, and the recipient C3H strain carries a donor B6 Mom1 allele. Hybrid progeny from congenic females mated to B6 Apc Min/+ males were analyzed. A single C3H Mom1 locus on the B6 background reduced small intestinal polyp numbers by 50% and colon polyp incidence by 66% compared to their susceptible B6 Mom1 S/S Apc Min/+ siblings. These findings show that the C3H genome contains a resistant Mom1 R locus. The reciprocal congenic line, which carries the susceptible B6 Mom1 S locus on the C3H background, reduced small intestinal polyp numbers by 80% and colon polyp incidence by 95% compared to B6 Mom1 S/S Apc Min/+ mice. These data demonstrate that unidentified modifiers in the C3H strain can suppress intestinal polyp multiplicity in Apc Min/+ mice, and act in the absence of a resistant Mom1 R locus.

Congenic mice reveal sex-specific genetic regulation of femoral structure and strength.

Genetic linkage studies in C3H/HeJ (C3H) and C57BL/6J (B6) mice identified several chromosomal locations or quantitative trait loci (QTL) linked to femoral volumetric bone mineral density (vBMD). From QTL identified on chromosomes (chr) 1, 4, 6, 13, and 18, five congenic mouse strains were developed. In each of these mice, genomic DNA from the QTL region of the donor C3H strain was transferred into the recipient B6 strain. Here we report the effects of donated C3H QTL on femoral structure, cortical vBMD and bending strength. Femoral structure was quantified by the polar moment of inertia (Ip) at the mid-diaphysis, which reflects the bending or torsional rigidity of the femur. Although the C3H progenitor mice have a smaller Ip than B6 progenitor mice, the congenic mice carrying the C3H segment at Chr 4 had significantly increased Ip in both males and females, giving these mice stronger femora. In female mice from the congenic Chr 1 strain, Ip was increased whereas male mice from the Chr 1 strain had smaller femoral cross-sections and significantly reduced Ip. This sex-specific effect on femoral structure was seen to a lesser extent in Chr 18 congenic mice. In addition, cortical vBMD was measured using peripheral quantitative computed tomography. Cortical vBMD was similar among most congenic strains except in Chr 6 congenic mice, where cortical vBMD was significantly less in females, but not in males. We conclude that (1) chromosomal QTL from C3H mice, which are genetically linked to total femoral vBMD, also regulate femoral structure; (2) the QTL on Chr 4 improves femoral structure and strength; (3) QTL on Chr 1 and 18 impart sex-specific effects on femoral structure; and (4) the QTL on Chr 6 imparts a sex-specific effect on cortical vBMD and femoral strength.

Differential CO(2)-induced c-fos gene expression in the nucleus tractus solitarii of inbred mouse strains.

Genetic determinants confer variation between inbred mouse strains with respect to the magnitude and pattern of ventilation during hypercapnic challenge. Specifically, inheritance patterns derived from low-responsive C3H/HeJ (C3) and high-responsive C57BL/6J (B6) mouse strains suggest that differential hypercapnic ventilatory sensitivity (HCVS) is controlled by two independent genes. The present study also tests whether differential neuronal activity in respiratory control regions of the brain is positively associated with strain variation in HCVS. With the use of whole body plethysmography, ventilation was assessed in C3 and B6 strains at baseline and during 30 min of hypercapnia (inspired CO(2) fraction = 0.15, inspired O(2) fraction = 0.21 in N(2)). Subsequently, in situ hybridization histochemistry was performed to determine changes in c-fos gene expression in the commissural subnucleus of the nucleus tractus solitarius (NTS). During hypercapnia, breathing frequency and tidal volume were significantly (P < 0.01) different between strains: C3 mice showed a slow, deep-breathing pattern relative to a rapid, shallow phenotype of B6 mice. CO(2)-induced increase in c-fos gene expression was significantly (P < 0.01) greater in NTS regions of B6 compared with C3 mice. In this genetic model of differential HCVS, the results suggest that a genomic basis for varied hypercapnic chemoreception or transduction confers greater afferent neuronal activity in the caudal NTS for high-responsive B6 mice compared with low-responsive C3 mice.

Genetic determinants of acute hypoxic ventilation: patterns of inheritance in mice.

Acutely lowering ambient O(2) tension increases ventilation in many mammalian species, including humans and mice. Inheritance patterns among kinships and between mouse strains suggest that a robust genetic influence determines individual hypoxic ventilatory responses (HVR). Here, we tested specific genetic hypotheses to describe the inheritance patterns of HVR phenotypes among two inbred mouse strains and their segregant and nonsegregant progeny. Using whole body plethysmography, we assessed the magnitude and pattern of ventilation in C3H/HeJ (C3) and C57BL/6J (B6) progenitor strains at baseline and during acute (3-5 min) hypoxic [mild hypercapnic hypoxia, inspired O(2) fraction (FI(O(2))) = 0.10] and normoxic (mild hypercapnic normoxia, FI(O(2)) = 0.21) inspirate challenges in mild hypercapnia (inspired CO(2) fraction = 0.03). First- and second-filial generations and two backcross progeny were also studied to assess response distributions of HVR phenotypes relative to the parental strains. Although the minute ventilation (VE) during hypoxia was comparable between the parental strains, breathing frequency (f) and tidal volume were significantly different; C3 mice demonstrated a slow, deep HVR relative to a rapid, shallow phenotype of B6 mice. The HVR profile in B6C3F(1)/J mice suggested that this offspring class represented a third phenotype, distinguishable from the parental strains. The distribution of HVR among backcross and intercross offspring suggested that the inheritance patterns for f and VE during mild hypercapnic hypoxia are consistent with models that incorporate two genetic determinants. These results further suggest that the quantitative genetic expression of alleles derived from C3 and B6 parental strains interact to significantly attenuate individual HVR in the first- and second-filial generations. In conclusion, the genetic control of HVR in this model was shown to exhibit a relatively simple genetic basis in terms of respiratory timing characteristics.

High colony forming capacity of primary cultured hepatocytes as a dominant trait in hepatocarcinogenesis-susceptible and resistant mouse strains

When hepatocytes isolated from mouse liver are cultivated in vitro, a small fraction of cells can survive, forming colonies, while most cells die within a few weeks. We compared the colony forming capacity of hepatocytes in three mouse strains; two strains susceptible for hepatocarcinogenesis, C3H/HeJ (C3) and DBA/2J mice (D2), and one resistant strain, C57BL/6J mice (B6). The colony forming capacity was about 3:2:1 for D2, C3 and B6 at the 4th week after start of culture, indicating that this capacity correlated with the susceptibility to tumor induction. When the colony forming capacity was compared in F1 hybrids between the three strains, the high colony forming capacity was dominant, again resembling the trait for hepatocarcinogenesis. In F1 hybrids between the two susceptible strains, the colony numbers were more than those of the parental strains, indicating the high colony forming capacity of the two susceptible strains to be additive. During 4 weeks of culture period, the cells continuously proliferated, but fairly large numbers of cells died, some showing characteristics of apoptosis and others of lysis. Although the proliferation rate was not different among the three strains until the 2 week time point, it was significantly lower in B6 than in C3 or D2 strains by the 4th week. On the other hand, the cell death rate was lower in D2 cells than in B6 or C3 cells after 2 weeks. These results indicate that the genetic background affects proliferation and death rates of cultured hepatocytes, which may be related to the different colony forming capacity of these three strains.

Circulating and skeletal insulin-like growth factor-I (IGF-i) concentrations in two inbred strains of mice with different bone mineral densities

Recent work has demonstrated differences in femoral bone mineral density between two common inbred strains of mice, C3H/HeJ (C3H) and C57BL/6J (B6), across a wide age range. To investigate one possible mechanism that could affect acquisition and maintenance of bone mass in mice, we studied circulatory and skeletal insulin-like growth factor-I (IGF-I) and femoral bone mineral density (F-BMD) by pQCT in C3H and B6 progenitor strains, as well as serum IGF-I obtained from matings between these two strains and mice bred from subsequent F 1 intercrosses (F 2). Serum IGF-I measured by radioimmunoassay was more than 35% higher in virgin progenitor C3H than virgin B6 at 1, 4, 8, and 10 months of age, and in 8-month-old C3H compared with B6 retired breeders ( p < 0.001). In the progenitors, there was also a strong correlation between serum IGF-I and serum alkaline phosphatase ( r = 0.51, p = 0.001). In the 4 month F 1 females IGF-I levels and F-BMD were intermediate between C3H and B6 progenitors. In contrast, groups of F 2 mice with the highest or lowest BMD also had the highest or lowest serum IGF-I ( p = 0.0001). IGF-I accounted for >35% of the variance in F-BMD among the F 2 mice. Conditioned media from newborn C3H calvarial cultures has higher concentrations of IGF-I than media from B6 cultures, and cell layer extracts from C3H calvariae exhibited greater alkaline phosphatase activity than cultures from B6 calvarial cells ( p < 0.0001). The skeletal content of IGF-I in C3H tibiae, femorae, and calvariae (6–14 weeks of age) was also significantly higher than IGF-I content in the same bones of the B6 mice ( p < 0.05). These data suggest that a possible mechanism for the difference in acquisition and maintenance of bone mass between these two inbred strains is related to systemic and skeletal IGF-I synthesis.

Genetic control of susceptibility to ozone-induced changes in mouse tracheal electrophysiology

Genetic factors influence the responses of humans and rodents to ozone (O3) inhalation. We previously demonstrated differential O3-induced decreases of tracheal potential (VT) in C57BL/6J (B6) and C3H/HeJ (C3) strain mice. To characterize the genetic basis of this strain-specific response, we measured VT in progeny of B6 and C3 strain mice and in six additional inbred strains of mice 6 h after O3 exposures (2 ppm x 3 h). First filial generation (F1) mice and second generation backcrosses with the resistant parent were uniformly resistant. The distribution of VT in second generation backcrosses with the susceptible parent resembled that of a population composed of resistant and susceptible mice in a 1:1 ratio. These data suggested simple autosomal recessive inheritance of susceptibility. However, overlapping distributions prevented statistical confirmation of that hypothesis. Strain screening revealed a susceptible phenotype in 129/J, A/J, B6, C3HeB/FeJ, and SJL/J and a resistant phenotype in AKR/J, C3, and CBA/J inbred mouse strains. Because this pattern of susceptibility to changes in VT differs from that of susceptibility to lung inflammation, the genetic factors that determine these two responses to acute O3 are not identical.

Differential control of ventilation among inbred strains of mice

The role genetic factors play in ventilatory control was examined by challenging eight inbred strains of mice to acute hypercapnia under normoxic and hypoxic conditions. Age-matched mice were exposed for 3-5 min to inspired gases of the following composition (FICO2:FIO2) 0.03:0.10, 2) 0.03:0.21, 3) 0.08:0.10, and 4) 0.08:0.21, with intermittent room air exposures. Breathing frequency (f) and tidal volume (VT) of unanesthetized, unrestrained mice were assessed by whole body plethysmography. During room air breathing, significant (P < 0.01) interstrain differences were noted in the pattern, but minute ventilation (VE) did not differ among the strains. Relative to room air, mild hypercapnia with hypoxia (0.03:0.10) significantly (P < 0.01) elevated VE in each strain, and the percent increase in VE of the DBA/2J strain was significantly (P < 0.05) greater than the other strains. The ventilatory response to these conditions was achieved primarily by a significant (P < 0.01) increase in f among the strains. During severely hypercapnic normoxia (0.08:0.21) and hypoxia (0.08:0.10), the increase in VE was significantly (P < 0.01) greatest in the C57BL/6J (B6) mice and least in the C3H/HeJ (C3) mice. The difference in hypercapnic VE between B6 and C3 strains was largely due to a significantly (P < 0.01) greater increase in VT by B6 mice. On the assumption that environmental factors were identical, these data suggest that genetic determinants govern interstrain variation in the magnitude and pattern of breathing during hypoxia and hypercapnia. Moreover, hypoxic and hypercapnic ventilatory responses appear to be influenced by different genetic mechanisms.