262 BRIEF REPORTS

tennial Fund o f the Bank of Sweden and the Swedish Cancer Society. The technical assistance of Miss DZINTRA TIDRIKS is gratefully acknowledged.

Institute of Physiological Botany, University of Uppsala, Sweden.

L i t e r a t u r e c i t e d

BENDER, I,. and SIVA SANKAR, D. V. 1968. Chromosome damage not found in leuko- cytes of children treated with LSD-25. Science 159: 749.

COHEN, M. M., MARINELLO, M. J. and BACK, N. 1967. Chromosomal damage in human leukocytes induced by lysergic acid diethylamide. - Ibid. 155: 1417- 1119.

COHEN, M. M. and MUKHERJEE, A. B. 1968. Meiotic chromosome damage induced by LSD-25. - Nature 219: 1072-1074.

GRACE, D., CARLSON, E. A. and GOODMAN, P. 1968. Drosophila rnelanognster treated with I S D : absence of mutation and chromosome breakage. - Science 1 G 1 : 0 4

IRWIN, S. and EGOZCUE, J. 1967. Chromosomal abnormalities in leukocytes from LSD-25 users. - Ibid. 157: 313-314.

KATO, R. 1967. Localization of "spontaneous" and Rous sarcoma virus-induced breakage in specific regions of the chromosomes of the Chinese hamster. - Hereditas 58: 221-247.

KIHLMAN, B. A. 1963. The effect of 5-halogenated deoxyuridines on the frequency of x-ray-induced chromosomal aberrations in Vicia faba. - Ibid. 49: 353-370.

I,OUGHMAN, w. D., SARCENT, T. W. and ISRAELSTAM, 1). M. 1967. Leukocytes of humans exposed to lysergic acid diethylamide: lack of chromosomal damage. - Science 158: 508-510.

MOORHEAD, P. S., NOWELL, P. C., MELLMAN, W. J., BATTIPS, D. M. and HUNGER- FORD, D. A. 1960. Chromosome preparations of leukocytes cultured from human peripheral blood. - Exp. Cell Res. 20: 613-616.

SKAKKEBEK, N. E., PHILIP, J. and RAFAELSEN, 0. J. 1968. LSD in mice: abnormali- ties in meiotic chromosomes. - Science 1GO: 12461218 .

SPARKES, R. S., MELNYK, J. and BOZZETTI, L. P. 1968. Chromosomal effect in vivo of exposure to lysergic acid diethylamide. - Ibid. $60: 1343-1315.

-696.

GOSTA ZETTERRERG: L y s e r g i c a c i d d i e t h y l a m i d e a n d m u t a t i o n . (Received October 8th, 1968)

There a r e several reports on the effects of lysergic acid diethylamide (LSUj on chromosomes of higher animals and plants (see STURELII) a n d KIHLMAN, 1969 for references) while the gene-mutagenic effect of I S D seems to have been Iittle studied. I n a recent study GRACE e f at. (1968) reported LSD ,to be non-mutagenic in Drosophila nielanoguster. The present investigation gave results which led to the conclusion that under the conditions employed LSD is not mutagenic to the test object, Opliiostoma multiannulatum, an asco- mycete.

The strains used in these experiments have a yeast-like growth a n d mor-

BRIEF REPORTS 263

phology. The wild type can grow on a medium containing a carbon source and simple inorganic salts plus two vitamins, thiamine and pyridoxine. Bio- chemical mutants can be induced with ultraviolet light (UV) , x-rays and many chemicals, e.g. alkylating agents. The most effective mutagens have given 2-4 per cent biochemical mutants (forward-mutants) among the survivors. The forward-mutation test in Ophiostoma was developed by FRIES (1948).

The methods used in the back-mutation tests have been described earlier (ZETTERRERG, 1960). The strain used here in back-mutation test is arginine- less and was induced with UV-irradiation. It reverts spontaneously to wild- type with a frequency that can be increased with UV-light. Revertants are selected on minimal medium.

The forward-mutution test. The cells were cultivated in liquid minimal medium a t 25' C. At the end of the logarithmic growth phase the cell suspen- sion was filtered through cotton wool, the cells collected by centrifugation and washed twice. Cells from 10 ml suspension were resuspended in 5 ml 1/15 M phosphate buffer pH 5.6 plus 5 ml of the LSD-solution. (The LSD used in these experiments was provided in ampoules, LSD-25, Sandoz AG, Basel, Lot. 67003, containing 0.1 mg LSD in 1 ml of a 0.7 per cent sodium chloride solution, the pH adjusted to 3.2 with 8.2 ,ug of tartaric acid). The final con- centration of LSD was 50 mg per liter. The cell suspension was placed on a shaker at 25* C for 13 hours. A control suspension was treated identically except that the LSD-solution was replaced by 5 ml of a solution containing NaCl and tartaric acid at the same concentration and acidity as in the LSD- solution.

After treatment the cells were washed twice, resuspended in 0.9 per cent NaC1-solution and plated on complete medium. During the treatment no growth of the cells was observed and the LSD-treatment had no killing effect.

After 6 days the small colonies formed on the plates were transferred to minimal agar. On this medium biochemical mutants would not grow. Of 1740 isolates none showed a decreased growth rate on minimal medium com- pared to the control. Thus the LSD-treatment was ineffective in induction of forward-mutations.

The back-mutation test. 2.5 ml of LSD-solution were added to each of 10 flasks containing 10 ml minimal medium+arginine (50 mg/liter) to give a final conc. of 20 mg LSD/liter. To 10 control flasks 2.5 ml of a NaCl+tartaric acid solution, pH 3.2 were added, instead of the LSD-solution. All flasks were inoculated with about 5 X lo5 cells of the arginine-less mutant and incubated on a shaker at 25' C for 3 days. During the incubation period the growth rate was a high in the I.SD culture\ as in the control cultures. Thus LSD is not toxic and does not affect the growth rate of Ophiostoma.

After 3 days the cultures had reached the end of the logarithmic phase of growth and contained about lo7 viable cells per ml. Ten ml from the flasks were washed twice in NaC1-solution and concentrated to a final volume of 1.0 (ml. On each of 5 plates of minimal medium 0.1 ml was plated to select for revertants to wild type. After proper dilutions 0.1 ml was plated on each of 5 plates of minimal medium+arginine as a viability test. The plates were incubated at 25'C and the number of colonies scored after 6 days. The frequences of revertants are shown in Table 1.

264 BRIEF REPORTS

TABLE 1. The frequencies o f revertants to wild type in 10 independent cul- turcs of an arginine-less strain o f Ophiostoma multiannnulatum grown in the presence of 20 mglliter of lysergic acid diethylamide (LSD) , and in 10 in-

dependent control cultures. The figures refer to the number of revertants per million cells.

Cultureno. 1 2 3 4 5 6 7 8 9 10 Mean

LSD 21.0 0.16 78.3 0.30 66.6 0.02 39.1 0.02 37.4 0.02 24.3 Control 256.0 0.34 0.01 96.7 0.02 0.18 0.25 0.94 0.0.5 9.60 36.4 t=0.1964 0.9>P>0.8

In the LSD series a s well as in the control series there is a great fluctuation in reversion frequency. This can be explained in the following way: A high reversion frequency is obtained when a reversion occurs a t a n early stage of growth giving rise t o many wild-type daughter cells before plating. A reverslion occuring late ,during growth will thus give a low reversion frequency. A sta- tistical analysis of the results, using the t-test, shows that LSD has no signi- ficant influence on the frequency of back-mutations in this strain of Ophiostoma.

Acknowledgements. - This study was supported by a grant from the Tri-Cen- tennial Fund of the Bank of Sweden. I thank bliss INGER NORDENHAMMAR for technical assistance.

Institute of Physiological Botany, University of Uppsala, Sweden.

L i t e r a t u r e c i t e d

FRIES, N . 1948. Viability and resistance of spontaneous mutations in Ophiostoniu representing different degrees of heterotrophy. - Physiol. Plant. I : 330-341.

GRACE, D., CARLSON, E. A. and GOODMAN, P. 1968. Drosophila melanogaster treated with LSD: absence of mutation and chroniosome breakage. - Science 161:601

STURELID, S. and KIHLMAN, B. A. 1969. Lysergic acid diethylamide and chromosome breakage. - Hereditas X: X-X.

ZETTERBERC, G. 1960. The mutagenic effect of 8-ethoxycaffein, caffein and di- methylsulfate in the Ophiostomu back-mutation test. - h i d . 46: 279-311.

-696.

KATARINA SULOVSKA, 1). LINDGREN, G . ERIKSSON and I,. EHRENHERG: T h e m u t a g e n i c e f f e c t o f l o w c o n c e n t r a t i o n s o f e t h y l e n e o x i d e i n a i r .

(Received October 16th, 1968)

Human beings a r e exposed to increasing numbers a n d amounts of chemicals, some of which have been shown to act radiomimetically. Their effects on m a n at low dose levels are of particular interest. However, so f a r it has experi-