Prenatal Effects of Elymoclavine Administration and Temperature Stress 1

PRENATAL EFFECTS OF ELYMOCLAVINE ADMINISTRATION AND TEMPERATURE STRESS 1

Weldon L. Witters, Robert A. Wilms and Ronald D. Hood 2

Ohio University, Atbens 45 701 Downloaded from https://academic.oup.com/jas/article/41/6/1700/4700896 by guest on 02 October 2021 SUMMARY al., 1965; Bailey et al., 1973. Some of the Elymoclavine, a toxic alkaloid produced by clavine (also called ergoline) compounds have Claviceps purpurea growing on feed grains and been claimed to be teratogenic or mutagenic forage grasses and produced by various bind- (Houston, 1969), some have oxytocic proper- weeds, was found to be embryocidal and ties (Brazeau, 1970), some inhibit prolactin teratogenic in mice when administered intra- release (Barkknecht and Ruterholz, 1974) and peritoneally at dose levels of 3, 30, or 60 mg/kg some seem to interfere with progestational on the 10th day of pregnancy. Elevated envi- activity (Finn and Mantle, 1969). Little or no ronmental temperatures (35 C or 40 C)tended research, however, has been published on the to exacerbate embryolethality. Maternal deaths possible teratogenic or mutagenic effects of were also noted at 30 C, 35 C and 40 C due to most of the individual constituent compounds. elymoclavine treatment. The fetal anomalies Previously published research with other produced consisted largely of vertebral defects compounds has indicated a temperature stress and fusion of the ribs. effect on pharmacologic activity (Shemano and (Key Words: Elymoclavine, Teratology, Alka- Nickerson, 1958; Fuhrman and Fuhrman, loid, Temperature Stress, Prenatal, Ergot.) 1961; Nomura et al., 1967). In preliminary experiments it was discovered that temperature INTRODUCTION may have a very important influence on the drug effects of ergot alkaloids, so a study was Elymoclavine, a close chemical relative of designed to test the prenatal effects of tempera- D-lysergic acid and lysergic acid diethylamide- ture and dosage of a representative alkaloid, 25 (Pelletier, 1970), has been found in ergot elymoclavine, utilizing the laboratory mouse. fungi (Claviceps purpurea) and in many of the morning-glories and bindweeks of the genus MATERIALS AND METHODS Ipomoea, family Convolvulaceae (Der Mardero- sian, 1967). Ergot has been reported growing An experiment of 4 • 4 factorial design was on grains such as rye, barley, wheat and millet, conducted using 245 albino mice of the inbred and on grasses such as perennial ryegrass. strain CCO:MLM-1 (ICR) 3. Food (Purina Lab Serious pathological effects have been reported Chow) and water were available ad libitum. in sheep and gilts grazing on either fresh or cut Treatment consisted of four levels of elymo- contaminated grass (Cunningham et al., 1944; clavine (0, 3, 30 and 60 mg/kg) tested at four Mantle, 1969; Bailey et al., 1973). Many of the temperatures (20, 30, 35 and 40 C). A high Convolvulaceae and infected ryegrasses are dosage of 60 mg/kg was chosen since in found in natural grazing areas and may be preliminary experiments an LDs0 of 67 mg/kg potentially dangerous to livestock consuming intraperitoneally (ip) was obtained for mice them (Gardner and Benneth, 1956; Gardiner et held at 20 C. Elymoclavine4 was dissolved in a .5% solution of tartaric acid, which was also used for treatment of controls. Two environmental chambers were em- Department of Zoology Paper. ployed. Chamber A s was kept at 20 + 2 C. 2 Department of Biology, University of Alabama. Chamber B 6 was maintained at 20 + 1 C, 30 + 1 3 Chordata Corporation, Ontario, New York. C, 35 + 1 C, or 40 + 1 C. Relative humidity 4Aldrich Chemical Company, Milwaukee, Wiscon- sin. ranged from 50 to 70% in both chambers. In s Hotpack model 1280. environmental chamber A, five females were 6Aminco model 4-139 HR. kept per cage along with one mature male. All 1700 JOURNAL OF ANIMAL SCIENCE, Vol. 41, No. 6, 1975 PRENATAL EFFECTS OF ELYMOCLAVINE 1701 mice experienced a 14-hr photoperiod (Raf- cervical dislocation. This early sacrifice facili- ferty, 1970). tated the clearing technique used in skeletal The female mice were checked at 8 am and examination. Resorptions and dead fetuses at 10 pm for vaginal plugs to determine the were noted at this time. Each litter was cleared time of pregnancy (Hood and Witters, 1972). and stained using a modified alizarin bone Those possessing a vaginal plug were transferred staining technique (Wilson and Warkany, 1967). to another cage. The day of observation of a All fetuses were later examined for gross vaginal plug at 8 am was recorded as the first skeletal anomalies, with emphasis on the dorsal day of gestation. Mice showing vaginal plugs at trunk (Gruneberg, 1952). 10 pm were recorded with the following day as Fisher's exact test for probability (Bradley, Downloaded from https://academic.oup.com/jas/article/41/6/1700/4700896 by guest on 02 October 2021 the first day of gestation. The 10th day of 1968) was used to test for significance. Data gestation was selected for the injections because were tested for dosage by temperature interac- it gave most effective results in pilot studies tion by use of analysis of variance 7 . (Rafferty, 1970). On that day, each pregnant mouse was injected ip with the appropriate RESULTS AND DISCUSSION dosage and transferred to environmental cham- The examination of fetuses on the 16th day ber B. After 24 hr the animals were removed of gestation revealed a number of resorptions in and returned to chamber A. several of the experimental groups (table 1). On the 16th day of a 19-day gestation One grossly abnormal fetus was observed in the period, all injected mice were sacrificed by group treated at 35 C with 30 mg/kg of elymoclavine. That fetus exhibited exencephaly and micrognathia, a condition which is known 7California Biomedical Programs, University of to appear "spontaneously" in the laboratory California, Los Angeles 90024. mouse.

Figure 1. Mouse fetus from mother treated with 30 Figure 2. Mouse fetus from control mother treated mg/kg at 35 C. Note fused ribs and defective verte- with tartaric acid carrier at 35 C. Note the even ribs brae. and vertebrae. 1702 WITTERS, WILMS AND HOOD Downloaded from https://academic.oup.com/jas/article/41/6/1700/4700896 by guest on 02 October 2021

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Preparation of the fetuses through skeletal more resorptions than was the Control treat- staining revealed a number of skeletal anomalies ment (P<.01). At the 40 C temperature, even (table 2). A number of treated fetuses exhibited the mice in the 3 mg/kg treatment group fused ribs and abnormal thoracic and lumbar exhibited a highly significant number of resorp- vertebrae (figure 1). The rib fusion was re- tions compared to control animals (P<".01). stricted to the sixth through 13th pairs and was In the three highest temperature groups, often adjacent to abnormal thoracic vertebrae. treatment with the higher dosages resulted in The vertebral anomalies involved the fusion of maternal deaths, particularly in the 40 C group vertebral arches and/or the dislocation of the (table 1). In the 40 C group, even the 3 mg/kg vertebral centra. No skeletal anomalies were treatment resulted in a significantly mortality Downloaded from https://academic.oup.com/jas/article/41/6/1700/4700896 by guest on 02 October 2021 found in the control mice (figure 2), rate for females compared to controls, while Frequencies of skeletal anomalies in the the 30 and 60 mg/kg treatments at that experimental fetuses appeared to be influenced temperature proved lethal to all the experimen- by dose as well as temperature (table 1). In the tal animals. From these data, it seems likely 20 C and 30 C groups, the difference in that the results of prenatal exposure to elymo- numbers of anomalies associated with the 60 clavine are both dose and temperature depen- mg/kg treated fetuses vs controls was highly dent. In use of chemicals that are teratogens, an significant (P<.01). At 35 C the 30 mg/kg increase in duration or intensity of treatment is treatment animals had a highly significant typically followed by results of greater fre- number of anomalies, and the 3 mg/kg treated quency or severity and are usually identified as fetuses in the 40 C group also differed from the more anomalies, total resorptions of fetuses, or controls (P<.01, table 1). The mice in the 3 finally maternal death (Green, 1966). There mg/kg treatment group at both 3 5 C and 40 C was a significant (P<.05) dosage by tempera- had significantly more resorptions (P<.01) than ture interaction for anomalies observed. As controls At 35 C animals receiving all levels of temperature increased the elymoclavine became elymoclavine were associated with significantly increasingly more biologically potent.

TABLE 2. ELYMOCLAV1NE-INDUCED FETAL ANOMALIES IN MICE: TEMPERATURE AND DOSAGE VS RESPONSE

Anomalies observed~t Dose Fetuses % vertebral % fused Temperature mg/kg examined defects ribs

20 C Control 131 0 o 3 129 0 o 30 82 2.4* 2.4* 60 105 11.4"* 11.4"* 30 C Control 132 0 o 3 92 0 o 30 135 .7* o 60 144 6.3** 2.8 35 C Control 144 0 o 3 152 0 o 30 124 11.3"* 5.6** 60 21 0 o 40 C Control 79 0 o 3 53 7.4** o 30 b 0 ... 60 b 0 ...

aExencephaly and micrognathia were observed in one fetus from a litter exposed to 30 mg/kg at 35 C. bAll treated animals died. *P<.05. * *P<.01. 1704 WITTERS, WILMS AND HOOD

Edwards (1971) believes that hyperthermia it may cause increased resorption in some cases. causes direct thermal damage to tissues. There Agroclavine also is said to block prolactin is adequate evidence from other research that release (Mantle, 1969). direct damage to cells and developing embryos Most of the research on biological activity can be produced by exposure to temperatures and potential teratogenicity of clavine alkaloids above the normal range. Stilwell (1957) re- has been conducted with lysergic acid diethyl- ported aberrations of mitosis in cell cultures of amide because of the interest in its drug abuse embryonic chicken hearts exposed to 42 C for potential. The evidence for lysergic acid dieth- 1 to 3 hr and Hasegawa (1955) found the ylamide as a teratogen or mutagen is highly ribonucleic content of toad embryos to be conflicting at the present time, with claims and Downloaded from https://academic.oup.com/jas/article/41/6/1700/4700896 by guest on 02 October 2021 decreased immediately after exposure for 10 counterclaims with regard to damage to the rain to elevated incubation temperatures (33, chromosomes, increased incidence of abortion 35 and 37 C). Alsop (1919) recorded a high and other dangers (Houston, 1969; Mantle, incidence of abnormalities particularly of the 1969; Long, 1972; Messier, 1973). Over 20 nervous system, in chicken embryos incubated related alkaloids have been isolated from lpo- at 39.4 to 42 C. In work on swine (Marple et moea and ergot fungus. Elymoclavine belongs al., 1974) it was found that as core tempera- to the large group of indole alkaloids called tures were increased from 27 C to 41.5 C there ergoline or clavine alkaloids. This group of were elevated levels of ACTH, adrenal corti- alkaloids has been found to be biologically coids and lactate as well as increased heart and active by Agurell and Ramstad (1962), Bark- respiration rates. knecht and Ruterholz (1974) and Grauwiler The indirect effects of hyperthermia that and Schoen (1973). could be involved are numerous and include inanition with loss of maternal weight, changes Campbell and Burfening (1972) found that in electrolyte concentrations, hypothermia, ingestion of ergot alkaloids lowered the preg- other general maternal reactions to "stress", nancy rate in gilts and mice, and that mice and damage to the placenta. ingesting these compounds had a significantly Other research has shown the importance of lower sustained implantation rate. Greatorex temperature on drug action. At some critical and MantIe (1974) found that rye ergot caused severe physical dysfunction in pregnant sheep. point, a rise in temperature may lead to an increase in toxicity (Fuhrman and Fuhrman, The pharmacology of elymoclavine has not 1961). The pharmacology of the ergot alkaloids been studied as completely as that of agroclav- may have also contributed to the increased ine or other ergot alkaloids. It seems likely lethality at higher temperature. Two ergot from observations in our laboratory that the alkaloids, ergotamine and LSD-25, produce a drug is a sympathomimetic, because of the rise in body temperature above 30 C but a effect on respiration rate and the occurrence of decrease below 30 C through some mechanism piloerection and convulsions at higher dosages. not yet understood (Shemano and Nickerson, The results of this investigation provide 1958). It is known that some of the clavine more insight into the effects of ergot alkaloids alkaloids have a sympathomimetic effect on the on mammals. They also imply that differences human at normal body temperatures. In mice, in environmental stresses may have influenced Nomura et al. (1967) found that environmental the often conflicting results obtained on fetal temperatures ranging between 25 C and 30 C development when rodents were treated with permit the stability of physiological functions. related compounds, such as LSD-25. Such Above and below that range, heart rate, respira- plants as morning-glory, the varied group often tion, body temperature, and blood pressure called bindweeds (which includes some morn- were affected. It is known that many of the ing-glories), leaves of the sweet potato (Ipo- ergoline compounds, such as elymoclavine, act rnoea batata), and ergot-infected grain and as vasoconstrictors or pressor substances in perennial ryegrass contain pharmacologically some dosages. Some of the ergot alkaloids are active drugs that need further study to assess used for the expulsion of afterbirth because of the potential dangers to animals that are ex- their ability to stimulate smooth muscle. Agro- posed to them. It appears that some undiag- clavine, a chemical closely related to elymo- nosed disease states of animals observed in the clavine, has been found to block progesterone past could have been due to ingestion of plants action, an effect which could also explain how containing ergoline alkaloids. PRENATAL EFFECTS OF ELYMOCLAVINE 1705

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