Maternal ethanol consumption: Effect on skeletal muscle development in guinea pig offspring

Alcohol, Vol. 4, pp. 11-16.Copyright©PergamonJournals Ltd., 1987. Printedin the U.S.A.

0741-8329/87$3.00 + .00

Maternal Ethanol Consumption: Effect on Skeletal Muscle Development in Guinea Pig Offspring CYNTHIA NYQUIST-BATrlE,*'

CYNTHIA UPHOFFt

A N D T H O M A S B. C O L E t

*School o f Basic Life Sciences, University of Missouri-Kansas City tCentral Faculty for Electron Microscopy, School of Medicine Oral Roberts University R e c e i v e d 22 July 1986 NYQUIST-BATTIE, C., C. UPHOFF AND T. B. COLE. Maternal ethanol consumption: Effect on skeletal muscle development in guinea pig offspring. ALCOHOL 4(1) 11-16, 1987.--The effect of ethanol on developing skeletal muscle was analyzed by examining the gastrocnemius muscle from newborn guinea pigs, exposed to ethanol during the second half of gestation. Electron microscopy revealed vacuolated sarcoplasmic reticula, enlarged lipid droplets, decreased glycogen and mitochondrial abnormalities in the skeletal muscle samples from the ethanol-exposed newborn guinea pigs. None of these abnormalities were seen in the newborn controls who were born to dams which consumed the same amount of calories as the ethanol-treated dams. The ethanol-associated abnormalities, seen in this study, are similar to those seen in ultrastructural examination of skeletal muscle from chronic alcoholics. Alcohol

Fetal Alcohol Syndrome

Skeletal muscle

Guinea pig

THE Fetal Alcohol Syndrome (FAS) is a common cause of birth defects. This syndrome is characterized by a growth deficiency, as well as central nervous system, skeletal, cardiac, craniofacial and genital abnormalities [8,16]. Alterations in muscle coordination and hypotonia may be present [1]. In certain cases of human FAS in which muscle biopsies were performed, myocyte development was deleteriously affected. Some of the reported problems were a failure of muscle plane closure which resulted in hernias [16], muscle hypotrophy and, at the ultrastructural level, sarcomeric dysplasia [1]. In some cases of FAS, congenital fiber disproportion consisting of irregular muscle fiber diameters has been reported [1]. Although these muscle pathologies are seen in humans born to alcoholics, little experimental work has been done to determine if similar changes are seen in animal models in which nutritional factors are controlled. Therefore, it is not clear if the muscle changes seen in FAS are due to exposure to ethanol alone, result from the malnourishment of alcoholics or are due to a combination of the two. An animal model of the teratogenic effects of ethanol on skeletal muscle would allow the investigation of the mechanism(s) of ethanol's deleterious effects, as well as would help determine what periods of skeletal muscle development are sensitive to ethanol. One prior study using rats did show that the weights of selected muscles were reduced in offspring of ethanol treated dams, although undernutrition may have played a role in these changes [6].

The present study examined the effect of maternal ethanol consumption during the second half of gestation on the development of the gastrocnemius muscle of the guinea pig. This period of in utero development corresponds to late second and third trimester, and early neonatal human development and avoids the period of embryogenesis. Guinea pigs have long gestational periods of 70 days and therefore are an ideal rodent in which to study the effects of ethanol on late fetal development, corresponding to that which occurs in human pregnancy. Human development that occurs in the third-trimester may be a most vulnerable period for ethanol insult at least for brain development [21]. Another advantage of using guinea pigs as an FAS model is that the guinea pig placenta has several morphological features in common with the human placenta [9]. In addition, several authors have suggested that the guinea pig is a more suitable model for ethanol studies than other rodents because of the similarities between guinea pig and man in ethanol absorption, hepatic metabolism and in the need for vitamin C [5, 17, 22, 23]. The present electron microscopic study revealed several abnormalities in the gastrocnemius muscle from the ethanol-exposed newborns that were not seen in muscle samples from the control newborns. These abnormalities were not the result of undernutrition or stress because control dams were fed the same amount of an isocaloric liquid diet and consumed similar amounts of food as the ethanol treatment group. These pathological Findings could be the

1Requests for reprints should be addressed to Cynthia Nyquist-Battie, School of Basic Life Sciences, University of Missouri-Kansas City, Kansas City, MO 64108.

ll

12

NYQUIST-BATTIE, UPHOFF AND COLE

FIG. 1. Typical micrograph of skeletal muscle from a newborn guinea pig whose dam was fed a sucrose solution twice daily as described in the Method section. (8400x)

TABLE 1 EFFECT OF ETHANOL EXPOSURE DURING THE SECOND HALF OF GESTATION ON THE OUTCOME OF PREGNANCY IN GUINEA PIGS

Gestational Length (Days) No. of Newborns/6 Dams Litter Size Newborn Weight (g) Newborn Length (cm)

Ethanol

Control

70.1 + 1.0 18 2-4 84.8 ± 4.3 10.7 -± 0.3

69.9 _+ 1.3 19 2-4 86.1 ± 4.9 11.6 +_ 0.2

No statistical differences were seen between the two groups in the above parameters (Student t-test).

manifestation of metabolic changes caused by e x p o s u r e to ethanol during d e v e l o p m e n t . METHOD Hartley strain guinea pigs, utilized in this study, were obtained from Murphy Breeding Laboratories (Plainfield, IN). F o u r t e e n female guinea pigs w e r e mated and their pregnancies w e r e determined by the presence of vaginal plugs (day 1 of pregnancy). Pregnant guinea pigs were housed in separate cages and were fed normal guinea pig c h o w with vitamins (ICN Vitamin Fortification mixture including vitamin C) added to their drinking water. On day 35 and for the duration of pregnancy, eight animals were fed from a medicine dropper, twice daily, a 30% ethanol solution m a d e

ETHANOL EXPOSURE I N U T E R O AND SKELETAL MUSCLE

13

FIG. 2. Micrograph of skeletal muscle from a newborn guinea pig whose dam was fed the ethanol solution (6 g/kg body weight) twice daily. Intermyofibrillar vacuoles are present and the amount of glycogen is reduced (8400×) up with Ensure liquid diet (Ross Laboratories) for a total of 6 g ethanol per kg body weight per day. The control group of six subjects received the same amount of solution, but with isocaloric replacement of ethanol by sucrose. Feedings were performed at 8 a.m. and 4 p.m. daily and took approximately 3 minutes per subject. Two of the pregnant animals were used to determine blood ethanol levels. These animals were placed under barbiturate anesthesia and blood was removed by cardiac puncture. Alcohol levels were determined using a kit from Sigma Chemical Co. (St. Louis, MO). At birth, the newborn guinea pigs were perfused through the left ventricles under barbiturate anesthesia with a warm phosphate-buffered saline solution containing 2.5% polyvinyl pyrrolidone (M.W. 40,000), heparin (1000

units/liter) and 0.5% procaine followed by an ice cold fLxative (1.5% glutaraldehyde, 1.5% paraformaldehyde, 0.25% polyvinyl pyrrolidone in 0.2 M sodium phosphate buffer pH 7.4). A volume of 25 ml of fLxative per 100 gram body weight was used. The gastrocnemius muscle was fixed for 12 hours in situ by placing the animal in a plastic bag and storing at 4°C. The upper lateral portion of the gastrocnemius from each left hind limb was removed and cut into 1 mm 3 blocks and kept in fucative for 24 hours. The blocks were postfixed in 1% osmium tetroxide with 1.5% ferricyanide and 3% sucrose, rinsed, dehydrated in ethanol and embedded in Epon 812-Araldite. Ultrathin sections were mounted on uncoated grids and stained with 5% uranyl acetate followed by Reynold's lead citrate solution. Sections were examined using a

14

N Y Q U I S T - B A T I ' I E , U P H O F F A N D COLE

FIG. 3. Legend same as Fig. 2 except for the presence of lipid droplets adjac to mitochondria. (16,500×)

random sampling technique. The sampling technique consisted of taking photographs of each upper right hand corner of the grid square (300 mesh). At least 20 photographs per block were taken. Twelve hundred sarcomeres were examined from animals in each group and were scored for pathologies. RESULTS The present regime of feeding pregnant guinea pigs with ethanol twice daily was resorted to after the liquid diet as the only source of nutrition failed. When the liquid diet was used as the only food, guinea pigs developed severe malnutrition and sometimes died because the animals would not ingest enough of the diet for proper nourishment. However, feeding animals the ethanol solution plus allowing them free access

to laboratory chow resulted in proper nutrition. Consumption of the ethanol solution twice daily by two pregnant guinea pigs resulted in blood ethanol levels of 179-200 mg% one hour post-feeding on day 40 of gestation. No significant differences were seen in food and water intake between pregnant dams fed the ethanol solution and those fed the sucrose solution. The food intake of the ethanol group was 23.2---6.1 g/day and was 23.1---7.6 g/day for the control group. The water intake was 75.0---9.4 and 76.6--.9.8 ml/day for the ethanol and control groups respectively. N o differences in the length of gestation and litter size were evident between groups (Table 1). Newborn weight and crown-torump length did not vary as a function of treatment, also shown in Table 1. Ultrastructural examination of the gastrocnemius muscle sample from one newborn in each litter was performed.

E T H A N O L EXPOSURE I N U T E R O AND S K E L E T A L MUSCLE Blocks were chosen randomly and were examined without knowledge of treatment. Figure 1 is a typical micrograph of a sample of gastrocnemius muscle from a newborn pup of the control group and shows normal muscle ultrastructure. No ultrastructural abnormalities were obvious in any of the six samples examined from this group. In contrast, sections of skeletal muscle obtained from the ethanol animals contained two major types of pathologies. The most obvious of these was the presence of intermyofibrillar vacuoles, which appear to be the vacuolization of the sarcoplasmic reticulum (SR) (Fig. 2). Intermyofibrillar vacuoles were observed in varying degrees in all samples from the ethanol-exposed newborns. Of the 1200 sarcomeres examined, approximately 81% had some degree of vacuolization. The examination of many sarcomeres with this pathology in different degrees suggested that the vacuolization began close to the Z-line and enlarged towards the M-band. Additionally, the interfibrillar areas with the vacuoles had less glycogen granules. A second type of pathology was found in the gastroenemius from the newborn guinea pigs of the ethanol group. A proportion of interfibrillar spaces of the muscle fibers were enlarged and contained large paramitochondrial lipid droplets (Fig. 3). These abnormal lipid droplets impinged upon adjacent mitochondria, which in turn were irregular in shape. These enlarged interfibrillar spaces with abnormal lipid droplets occurred in different sarcomeres than the vacuolization of the SR. The enlarged lipid droplets were obvious in samples from all six newborns exposed to ethanol in utero, and occurred in 16% of the sarcomeres examined. DISCUSSION

In the present study, the effect of ethanol on skeletal muscle development was investigated in the guinea pig. Since their period of gestation is longer than most rodents, the effect of ethanol in utero on advanced stages of skeletal muscle development could be examined. In this model of FAS, pregnant guinea pigs consumed 6 g of ethanol/kg body weight from day 35 of pregnancy until birth. They still consumed the same amount of food and water as the control group, thereby negating any effects due to undernutrition. Pathologies, detectable by electron microscopy and attributable to ethanol, were present in the sampled skeletal muscle. These included a vacuolated sarcoplasmic reticulum, an increased size of lipid droplets, irregularly shaped mitochondria and decreased interfibrillar glycogen. Interestingly, the increased fatty deposits and the SR vacuolization seen in this study occurred in separate sarcomeres and possibly in separate muscle fiber types. Guinea pig gastrocnemius muscle contains three types of fibers: fast twitch-glycolytic (50%), fast twitch-glycolytic and oxidative (38.6%) and slow twitch-oxidative 02%) [2]. Given the fact that the gastrocnemius has predominately fast twitch fibers and that the slow twitch fibers contain mitochondria and lipid droplets in much higher amounts than the fast twitch fibers [2], it would seem logical to conclude from the present data that the ethanol-induced SR vacuolization occurs in the fast twitch fibers while the ethanol-induced enlarged lipid droplets occur in the slow twitch fibers. This separation of effects would reflect a different response to ethanol by fast and slow twitch fiber types. A selective atrophy of anaerobic glycolytic (fast twitch) fibers has been reported in alcoholics [4,12], although it has not been found by all investigators [3,10]. It could be hypothesized that the different fiber types have different responses to ethanol because of their different metabolic compositions.

15

The ethanol-treated newborn guinea pigs of this study also exhibited central nervous system damage evident on histological examination [11]. This damage included a reduction in olfactory bulb size due to reduced neuronal growth, problems with neuronal migration and other neuronal abnormalities. These animals did not show alterations in craniofacial features analogous to those reported by Sulik et al. [18] in mice probably because embryogenesis was complete by the time of ethanol treatment in the present study. Similar ultrastructural pathologies to those reported in this paper in developing animals have been observed in skeletal muscle of adult alcoholics and in skeletal muscle of adult laboratory animals exposed to ethanol [3, 4, 14]. Therefore, maturational state once embryogenesis is complete may not play a role in the type of damage ethanol produces in striated muscle. It would appear however, that shorter periods of exposure to ethanol can, in younger animals, cause the ultrastructural pathologies that need longer periods of ethanol exposure to develop in the adult animal. It is not clear from the present study whether these changes are transient or will remain throughout adulthood. In our work on the effect of ethanol on the development of mouse heart, the changes seen at birth due to alcohol exposure in utero did not persist [19,20]. The uitrastructural changes we observed in skeletal muscle after ethanol exposure in utero differ from those observed in FAS children [1]. The children used in that study were chosen because of their advanced muscular weakness and exhibited a very advanced stage of myocyte breakdown. The reason for the differences between the two studies could be because of a synergism between other factors and ethanol in the human study or could be because of longer periods of ethanol exposure and higher doses in the mothers of these children. Alternatively, human muscle may be more sensitive than that of guinea pig to the effects of ethanol. The causes of the ethanol-induced changes in skeletal muscle seen in the present study are not known. Changes in ion handling and, in particular, that of calcium by the sarcoplasmic reticulum could cause SR vacuolization [13]. A change in the Na+K ÷ ATPase activity of skeletal muscle after ethanol exposure and the inhibition by ethanol of the sodium pump may also play a role [7]. Enlarged intermyofibrillar lipid droplets similar to what was found in this study are present in individuals with abnormally low muscle carnitine levels [15]. A deficiency in carnitine may cause decreased long chain fatty acid oxidation and in turn, increased lipid deposits. Possibly, ethanol exposure of guinea pigs in utero causes changes in carnitine levels or in some other manner alters fetal long chain fatty acid oxidation with a resultant increase in lipid stores. It can be concluded from this work that ethanol exposure in the second half of gestation can cause changes in skeletal muscle organelles similar to changes seen in adult skeletal muscle after chronic ethanol exposure. This suggests that ethanol exposure, at least during the later stages of maturation, may have the same effects on developing muscle as it does on mature muscle. However, developing muscle may be more sensitive to ethanol and shorter periods of exposure than those needed in adult muscle may result in damage. This investigation also shows the usefulness of the guinea pig for FAS studies, in which the effects of ethanol on human third-trimester equivalent development are to be investigated.

16

NYQUIST-BATTIE,

UPHOFF AND COLE

ACKNOWLEDGEMENTS This research was supported by NIAAA Grant IR03AA05976 and by a Grant-In-Aid from the American Heart Association, both to C.N.B.

REFERENCES 1. Adickes, E. and R. Shuman. Fetal alcohol myopathy. Pediatr Pathol 1: 36%384, 1983. 2. Close, R. T. Dynamic properties of mammalian skeletal muscle. Physiol Rev 52: 12%147, 1972. 3. Del Villar Negro, A., J. Merino Angulo and J. Rivera-Pomar. Skeletal muscle changes in chronic alcoholic patients. Acta Neurol Scand 70: 185-196, 1984. 4. Hailer, R. G. and J. P. Knochel. Skeletal muscle disease in alcoholism. Med Clin North Am 68: 91-103, 1984. 5. Hanson, R. W. The choice of animal species for studies of metabolic regulation. Nutr Rev 32: 1-8, 1974. 6. Ihemelandu, E. C. Effect of maternal alcohol consumption on pre- and post-natal muscle development of mice. Growth 45: 35-43, 1984. 7. Israel, Y. Cellular effects of alcohol: A review../Stud Alcohol 31: 23%316, 1970. 8. Jones, K. L. and D. W. Smith. Recognition of the Fetal Alcohol Syndrome in infancy. Lancet 2: 99%1001, 1973. 9. Kaufmann, P. Electron microscopy of the guinea pig placental membranes. Placenta [Suppl] 2: 3-10, 1981. 10. Martin, J. B., J. W. Craig, R. E. Eckel and L. Munger. Hypokalemic myopathy in chronic alcoholism. Neurology 21: 1160-1168, 1971. 11. Nyquist-Battie, C. Olfactory bulb development in guinea pigs exposed to ethanol during the latter half of gestation. Soc Neurosci Abstr 11: 524, 1985. 12. Peters, T. J., F. Martin and K. Ward. Chronic alcoholic skeletal myopathy--common and reversible. Alcohol 2: 485-489, 1985. 13. Rubin, E. Alcoholic mvopathy in heart and skeletal muscle. N Engl J Med 301: 28-33, 1978. 14. Rubin, E., A. M. Katz, C. S. Lieber, E. P. Stein and S. Puszkin. Muscle damage produced by chronic alcohol consumption. Am J Pathol 83: 499-516, 1976.

15. Schochet, S. S. and P. W. Lambert. Diagnostic Electron Microscopy, edited by B. B. Trump. New York: Wiley Medical Publications, 1978, p. 223. 16. Streissguth, A., S. Landesman-Dwyer, J. C. Martin and D. W. Smith. Teratogenic effects of alcohol in humans and laboratory animals. Science 204: 353-361, 1980. 17. Strubelt, O., C. P. Sigers and H. Breining. Comparative study of the absorption, elimination and acute hepatoxic action of ethanol in guinea pigs and rats. Arch Toxicol 32: 83-95, 1974. 18. Sulik, K. K. and M. C. Johnston. Sequence of developmental alternations following acute ethanol exposure in mice: Craniofacial features of the Fetal Alchol Syndrome. Am J Anat 166: 257-264, 1983. 19. Uphoff, C. and C. Nyquist-Battie. Cardiac muscle development in mice exposed to ethanol in utero. Teratology 30: 11%124, 1984. 20. Uphoff, C. and C. Nyquist-Battie. Effects of alcohol and undernutrition on mouse myocardial development. Anat Rec 211: 366, 1985. 21. West, J. R., K. M. Hamre and M. D. Cassell. Effects of ethanol exposure during the third trimester equivalent on neuron number in rat hippocampus and dentate gyrus. Alcohol: Clin Exp Res 10: 140-14% 1986. 22. Yunice, A. A., J. M. Hus, A. Fahmy and S. Henry. Ethanolascorbate interrelationships in acute and chronic alcoholism in the guinea pig. Proc Soc Exp Biol Med 177: 262-271, 1984. 23. Zahlter, R. N., M. E. Nejteck and J. C. Jacobsen. Ethanol metabolism in guinea pigs: Ethanol oxidation and its effects on NAD/NADH ratios, oxygen consumption and ketogenesis in isolated hepatocyte of fed and fasted animals. Arch Biochem Biophys 213: 200-231, 1982.