Compared to sucrose, previous consumption of fructose and glucose monosaccharides reduces survival and fitness of female mice.

BACKGROUND: Intake of added sugar has been shown to correlate with many human metabolic diseases, and rodent models have characterized numerous aspects of the resulting disease phenotypes. However, there is a controversy about whether differential health effects occur because of the consumption of either of the two common types of added sugar-high-fructose corn syrup (fructose and glucose monosaccharides; F/G) or table sugar (sucrose, a fructose and glucose disaccharide). OBJECTIVES: We tested the equivalence of sucrose- vs. F/G-containing diets on mouse (Mus musculus) longevity, reproductive success, and social dominance. METHODS: We fed wild-derived mice, outbred mice descended from wild-caught ancestors, a diet in which 25% of the calories came from either an equal ratio of F/G or an isocaloric amount of sucrose (both diets had 63% of total calories as carbohydrates). Exposure lasted 40 wk, starting at weaning (21 d of age), and then mice (104 females and 56 males) were released into organismal performances assays-seminatural enclosures where mice competed for territories, resources, and mates for 32 wk. Within enclosures all mice consumed the F/G diet. RESULTS: Females initially fed the F/G diet experienced a mortality rate 1.9 times the rate (P = 0.012) and produced 26.4% fewer offspring than females initially fed sucrose (P = 0.001). This reproductive deficiency was present before mortality differences, suggesting the F/G diet was causing physiologic performance deficits prior to mortality. No differential patterns in survival, reproduction, or social dominance were observed in males, indicating a sex-specific outcome of exposure. CONCLUSION: This study provides experimental evidence that the consumption of human-relevant levels of F/G is more deleterious than an isocaloric amount of sucrose for key organism-level health measures in female mice.

Mouse fitness measures reveal incomplete functional redundancy of Hox paralogous group 1 proteins.

Here we assess the fitness consequences of the replacement of the Hoxa1 coding region with its paralog Hoxb1 in mice (Mus musculus) residing in semi-natural enclosures. Previously, this Hoxa1B1 swap was reported as resulting in no discernible embryonic or physiological phenotype (i.e., functionally redundant), despite the 51% amino acid sequence differences between these two Hox proteins. Within heterozygous breeding cages no differences in litter size nor deviations from Mendelian genotypic expectations were observed in the outbred progeny; however, within semi-natural population enclosures mice homozygous for the Hoxa1B1 swap were out-reproduced by controls resulting in the mutant allele being only 87.5% as frequent as the control in offspring born within enclosures. Specifically, Hoxa1B1 founders produced only 77.9% as many offspring relative to controls, as measured by homozygous pups, and a 22.1% deficiency of heterozygous offspring was also observed. These data suggest that Hoxa1 and Hoxb1 have diverged in function through either sub- or neo-functionalization and that the HoxA1 and HoxB1 proteins are not mutually interchangeable when expressed from the Hoxa1 locus. The fitness assays conducted under naturalistic conditions in this study have provided an ultimate-level assessment of the postulated equivalence of competing alleles. Characterization of these differences has provided greater understanding of the forces shaping the maintenance and diversifications of Hox genes as well as other paralogous genes. This fitness assay approach can be applied to any genetic manipulation and often provides the most sensitive way to detect functional differences.

Sexual selection constrains the body mass of male but not female mice.

Sexual size dimorphism results when female and male body size is influenced differently by natural and sexual selection. Typically, in polygynous species larger male body size is thought to be favored in competition for mates and constraints on maximal body size are due to countervailing natural selection on either sex; however, it has been postulated that sexual selection itself may result in stabilizing selection at an optimal mass. Here we test this hypothesis by retrospectively assessing the influence of body mass, one metric of body size, on the fitness of 113 wild-derived house mice (Mus musculus) residing within ten replicate semi-natural enclosures from previous studies conducted by our laboratory. Enclosures possess similar levels of sexual selection, but relaxed natural selection, relative to natural systems. Heavier females produced more offspring, while males of intermediate mass had the highest fitness. Female results suggest that some aspect of natural selection, absent from enclosures, acts to decrease their body mass, while the upper and lower boundaries of male mass are constrained by sexual selection.

Rofecoxib-Induced Deleterious Effects Escape Detection by Organismal Performance Assays.

BACKGROUND: Organismal Performance Assays (OPAs) are a unique toxicity quantification method used to assess the safety of potentially toxic compounds, such as pharmaceuticals. OPAs utilize genetically diverse wild mice (Mus musculus) housed in semi-natural enclosures wherein exposed individuals compete directly with controls for resources. Previously, OPAs have been successful in detecting adverse effects in mice that were exposed to paroxetine. Here, we further test OPAs utility in pharmaceutical safety assessment by testing OPAs with rofecoxib, a drug with known adverse effects in humans. METHODS: We exposed mice to rofecoxib (~37.5 mg/kg/day) during gestation and into early adulthood. Exposure ceased when individuals were released into enclosures. Five independent populations were established and rofecoxib-exposed individuals (n = 58) competed directly with control individuals (n = 58) over 28 weeks. Organismal performance was determined by quantifying reproduction, survival and male competitive ability. RESULTS: In enclosures, rofecoxib-exposed males had equal reproduction, survival and competitive ability. Rofecoxib-exposed females had equal survival compared to controls but experienced 40% higher reproductive output. CONCLUSIONS: The adverse health effects of rofecoxib seen in humans escaped detection by OPAs, just as they had during traditional preclinical assays. These results may be explained by the exposure design (in enclosures, all animals were on the control diet), the relatively short duration of exposure, species differences, or because the health benefits of the drug negated the side effects. Similarly to numerous assays used in preclinical trials, OPAs cannot reveal all maladies, despite their demonstrated sensitivity in detecting cryptic toxicity from numerous exposures.

Quantification of cerivastatin toxicity supports organismal performance assays as an effective tool during pharmaceutical safety assessment.

A major problem in pharmaceutical development is that adverse effects remain undetected during preclinical and clinical trials, but are later revealed after market release when prescribed to many patients. We have developed a fitness assay known as the organismal performance assay (OPA), which evaluates individual performance by utilizing outbred wild mice (Mus musculus) that are assigned to an exposed or control group, which compete against each other for resources within semi-natural enclosures. Performance measurements included reproductive success, survival, and male competitive ability. Our aim was to utilize cerivastatin (Baycol(®), Bayer), a pharmaceutical with known adverse effects, as a positive control to assess OPAs as a potential tool for evaluating the safety of compounds during preclinical trials. Mice were exposed to cerivastatin (~4.5 mg/kg/day) into early adulthood. Exposure ceased and animals were released into semi-natural enclosures. Within enclosures, cerivastatin-exposed females had 25% fewer offspring and cerivastatin-exposed males had 10% less body mass, occupied 63% fewer territories, sired 41% fewer offspring, and experienced a threefold increase in mortality when compared to controls. OPAs detected several cerivastatin-induced adverse effects indicating that fitness assays, commonly used in ecology and evolutionary biology, could be useful as an additional tool in safety testing during pharmaceutical development.

Fitness Assays Reveal Incomplete Functional Redundancy of the HoxA1 and HoxB1 Paralogs of Mice.

Gene targeting techniques have led to the phenotypic characterization of numerous genes; however, many genes show minimal to no phenotypic consequences when disrupted, despite many having highly conserved sequences. The standard explanation for these findings is functional redundancy. A competing hypothesis is that these genes have important ecological functions in natural environments that are not needed under laboratory settings. Here we discriminate between these hypotheses by competing mice (Mus musculus) whose Hoxb1 gene has been replaced by Hoxa1, its highly conserved paralog, against matched wild-type controls in seminatural enclosures. This Hoxb1(A1) swap was reported as a genetic manipulation resulting in no discernible embryonic or physiological phenotype under standard laboratory tests. We observed a transient decline in first litter size for Hoxb1(A1) homozygous mice in breeding cages, but their fitness was consistently and more dramatically reduced when competing against controls within seminatural populations. Specifically, males homozygous for the Hoxb1(A1) swap acquired 10.6% fewer territories and the frequency of the Hoxb1(A1) allele decreased from 0.500 in population founders to 0.419 in their offspring. The decrease in Hoxb1(A1) frequency corresponded with a deficiency of both Hoxb1(A1) homozygous and heterozygous offspring. These data suggest that Hoxb1 and Hoxa1 are more phenotypically divergent than previously reported and support that sub- and/or neofunctionalization has occurred in these paralogous genes leading to a divergence of gene function and incomplete redundancy. Furthermore, this study highlights the importance of obtaining fitness measures of mutants in ecologically relevant conditions to better understand gene function and evolution.

Low-dose paroxetine exposure causes lifetime declines in male mouse body weight, reproduction and competitive ability as measured by the novel organismal performance assay.

Paroxetine is a selective serotonin reuptake inhibitor (SSRI) that is currently available on the market and is suspected of causing congenital malformations in babies born to mothers who take the drug during the first trimester of pregnancy. We utilized organismal performance assays (OPAs), a novel toxicity assessment method, to assess the safety of paroxetine during pregnancy in a rodent model. OPAs utilize genetically diverse wild mice (Mus musculus) to evaluate competitive performance between experimental and control animals as they compete among each other for limited resources in semi-natural enclosures. Performance measures included reproductive success, male competitive ability and survivorship. Paroxetine-exposed males weighed 13% less, had 44% fewer offspring, dominated 53% fewer territories and experienced a 2.5-fold increased trend in mortality, when compared with controls. Paroxetine-exposed females had 65% fewer offspring early in the study, but rebounded at later time points, presumably, because they were no longer exposed to paroxetine. In cages, paroxetine-exposed breeders took 2.3 times longer to produce their first litter and pups of both sexes experienced reduced weight when compared with controls. Low-dose paroxetine-induced health declines detected in this study that were undetected in preclinical trials with doses 2.5-8 times higher than human therapeutic doses. These data indicate that OPAs detect phenotypic adversity and provide unique information that could be useful towards safety testing during pharmaceutical development.

Infection-dependent phenotypes in MHC-congenic mice are not due to MHC: can we trust congenic animals?

BACKGROUND: Congenic strains of mice are assumed to differ only at a single gene or region of the genome. These mice have great importance in evaluating the function of genes. However, their utility depends on the maintenance of this true congenic nature. Although, accumulating evidence suggests that congenic strains suffer genetic divergence that could compromise interpretation of experimental results, this problem is usually ignored. During coinfection studies with Salmonella typhimurium and Theiler's murine encephalomyelitis virus (TMEV) in major histocompatibility complex (MHC)-congenic mice, we conducted the proper F2 controls and discovered significant differences between these F2 animals and MHC-genotype-matched P0 and F1 animals in weight gain and pathogen load. To systematically evaluate the apparent non-MHC differences in these mice, we infected all three generations (P0, F1 and F2) for 5 MHC genotypes (b/b, b/q and q/q as well as d/d, d/q, and q/q) with Salmonella and TMEV. RESULTS: Infected P0 MHC q/q congenic homozygotes lost significantly more weight (p = 0.02) and had significantly higher Salmonella (p < 0.01) and TMEV (p = 0.02) titers than the infected F2 q/q homozygotes. Neither weight nor pathogen load differences were present in sham-infected controls. CONCLUSIONS: These data suggest that these strains differ for genes other than those in the MHC congenic region. The most likely explanation is that deleterious recessive mutations affecting response to infection have accumulated in the more than 40 years that this B10.Q-H-2q MHC-congenic strain has been separated from its B10-H-2b parental strain. During typical experiments with congenic strains, the phenotypes of these accumulated mutations will be falsely ascribed to the congenic gene(s). This problem likely affects any strains separated for appreciable time and while usually ignored, can be avoided with the use of F2 segregants.

Human-relevant levels of added sugar consumption increase female mortality and lower male fitness in mice.

Consumption of added sugar has increased over recent decades and is correlated with numerous diseases. Rodent models have elucidated mechanisms of toxicity, but only at concentrations beyond typical human exposure. Here we show that comparatively low levels of added sugar consumption have substantial negative effects on mouse survival, competitive ability, and reproduction. Using Organismal Performance Assays--in which mice fed human-relevant concentrations of added sugar (25% kcal from a mixture of fructose and glucose, modeling high fructose corn syrup) and control mice compete in seminatural enclosures for territories, resources and mates--we demonstrate that fructose/glucose-fed females experience a twofold increase in mortality while fructose/glucose-fed males control 26% fewer territories and produce 25% less offspring. These findings represent the lowest level of sugar consumption shown to adversely affect mammalian health. Clinical defects of fructose/glucose-fed mice were decreased glucose clearance and increased fasting cholesterol. Our data highlight that physiological adversity can exist when clinical disruptions are minor, and suggest that Organismal Performance Assays represent a promising technique for unmasking negative effects of toxicants.