Embryonic arsenic exposure reduces the number of muscle fibers in killifish (Fundulus heteroclitus).

Arsenic is a contaminant of drinking water and has been correlated with adverse developmental outcomes such as low birth weight, reduced weight gain, and altered locomotor activity. Previous research has shown that killifish (Fundulus heteroclitus) exposed to high arsenic levels during embryogenesis had smaller muscle fiber diameters. The current study was designed to determine whether changes in muscle fibers persisted, were exacerbated, or resolved over time. Killifish embryos were exposed to 0-5 ppm arsenite and, upon hatching, were transferred into either clean water or continued receiving the same exposure to arsenic for up to 16 weeks. Arsenic significantly decreased the weight of both embryonic-only exposed juveniles and continuously exposed juveniles between 4 and 16 weeks of development at concentrations as low as 0.8 ppm. Although arsenite exposure increased the percentage of small diameter fibers during the early weeks, fiber diameters returned to control levels in the embryonic-only exposed fish. However, muscle fiber density was still reduced to 85.7%, 80.3%, and 73.8% of control for the 0.8, 2, and 5 ppm embryonic-only exposure groups, respectively, even after 16 weeks of development. These results indicate that while continuous exposure to arsenic may alter the size of muscle fibers, embryonic-only exposure to arsenic has lasting effects on the number of muscle fibers formed.

Diversity of limb-bone safety factors for locomotion in terrestrial vertebrates: evolution and mixed chains.

During locomotion over land, vertebrates' limb bones are exposed to loads. Like most biological structures, limb bones have a capacity to withstand greater loads than they usually experience, termed a safety factor (SF). How diverse are limb-bone SFs, and what factors correlate with such variation? We have examined these questions from two perspectives. First, we evaluated locomotor SF for the femur in diverse lineages, including salamanders, frogs, turtles, lizards, crocodilians, and marsupials (opossums). Comparisons with values for hind-limb elements in running birds and eutherian mammals indicate phylogenetic diversity in limb-bone SF. A high SF (∼7) is primitive for tetrapods, but low magnitudes of load and elevated strength of bones contribute to different degrees across lineages; moreover, birds and eutherians appear to have evolved lower SFs independently. Second, we tested the hypothesis that SFs would be similar across limb bones within a taxon by comparing data from the humerus and femur of alligators. Both in bending and in torsion, we found a higher SF for the humerus than for the femur. Such a "mixed chain" of different SFs across elements has been predicted if bones have differing variabilities in load, different costs to maintain, or high SF values in general. Although variability in load is similar for the humerus and femur, a high SF may be less costly for the humerus because it is smaller than the femur. The high SFs of alligators also might facilitate differences in SF among their limb bones. Beyond these specific findings, however, a more general implication of our results is that evaluations of the diversity of limb-bone SFs can provide important perspective to direct future research. In particular, more complete understanding of variation in SF could provide insight into factors that promoted the evolutionary radiation of terrestrial locomotor function in vertebrates.

Arsenic exposure to killifish during embryogenesis alters muscle development.

Epidemiological studies have correlated arsenic exposure in drinking water with adverse developmental outcomes such as stillbirths, spontaneous abortions, neonatal mortality, low birth weight, delays in the use of musculature, and altered locomotor activity. Killifish (Fundulus heteroclitus) were used as a model to help to determine the mechanisms by which arsenic could impact development. Killifish embryos were exposed to three different sodium arsenite concentrations and were collected at 32 h post-fertilization (hpf), 42 hpf, 168 hpf, or < 24 h post-hatch. A killifish oligo microarray was developed and used to examine gene expression changes between control and 25-ppm arsenic-exposed hatchlings. With artificial neural network analysis of the transcriptomic data, accurate prediction of each group (control vs. arsenic-exposed embryos) was obtained using a small subset of only 332 genes. The genes differentially expressed include those involved in cell cycle, development, ubiquitination, and the musculature. Several of the genes involved in cell cycle regulation and muscle formation, such as fetuin B, cyclin D-binding protein 1, and CapZ, were differentially expressed in the embryos in a time- and dose-dependent manner. Examining muscle structure in the hatchlings showed that arsenic exposure during embryogenesis significantly reduces the average muscle fiber size, which is coupled with a significant 2.1- and 1.6-fold upregulation of skeletal myosin light and heavy chains, respectively. These findings collectively indicate that arsenic exposure during embryogenesis can initiate molecular changes that appear to lead to aberrant muscle formation.