In Vitro Studies

Cohen, Marinello and Back (22) added LSD to cultured human leucocytes obtained from two healthy individuals. They used five concentrations ranging from 0.001 to 10.0 micrograms of LSD per cubic centimeter (cc), and the time of exposure was 4, 24, and 48 hours. The incidence of chromosome breaks for treated cells was at least twice that of control cells for all treatments, except at the lowest concentration and time (0.001 micrograms of LSD per cc for four hours) where no difference existed between treated and control cells. There was no simple linear relationship between the frequency of these aberrations and the LSD dosage or duration of exposure. Id a later study, Cohen, Hirschhorn and Frosch (20) described the results of a larger study in which they used peripheral leucocyte cultures from six normal, healthy persons; the concentrations of LSD and the times of exposure were the same as in the original study. They found a significant inhibition of cellular division (mitosis) on addition of the drug in any concentration. The suppression of mitosis was directly proportional to the duration of exposure. The lowest frequency of chromosomal breakage among the controls was 3.9 percent of cells; among the treated cultures, the lowest frequency was almost twice the control (7.7 percent) and ranged to over four times the control value (17.5 percent).

In 1968, Jarvik et al. (63) tried to replicate some of the in vitro experiments of Cohen's group. In addition to LSD, they used as testing substances ergonovine (a drug commonly used in obstetric practice), aspirin, and streptonigrine. They found a higher incidence of chromosome breaks in the LSD samples (10.2 percent with the range 0.0-15.0) as compared to the control samples (5.2 percent with the range from 0.0-9.0). They found, however, approximately the same breakage rate with aspirin (10.0 percent) and ergonovine (9.6 percent). The concentration of LSD in blood used in this study approximates the level reached one to four hours after injection of 1,000 micrograms of LSD. On the other hand, the level of aspirin used was considerably below the common therapeutic level. Streptonigrine, a substance with a well-known dramatic effect on the chromosomes, induced chromosome breakage in 35 percent of the examined cells. It is interesting to note that two of the eight cases described in this paper did not respond to LSD with an increase in chromosome breaks.

Corey el al. (24) performed an in vitro study in ten individuals; 1 microgram per cc of LSD was added to the culture during the last twenty-four hours of incubation. The authors found an increase in chromosome breaks in all ten subjects. Although the in vitro concentration of LSD was much greater than any known comparable ingested dosage, the mean increase of 4.65 breaks per 100 cells was small compared to the range of frequencies (0.0-15.2) observed in the untreated cultures.

In this connection it is interesting to mention that Singh, Kalia and Jain (92) found an increased incidence of chromosome breakage in the cells of barley root as a result of exposure to LSD in the concentration 25 micrograms per cc. On the other hand, MacKenzie and Stone (73) reported negative results of experiments on lymphocytes, hamster fibroblasts and on the plant Vicia faba.

The above-mentioned findings of structural changes in chromosomes following LSD administration became the basis of speculations concerning the possible influence of this drug on genetic mutations, fetal development and malignancy. In the atmosphere of national hysteria then existing, the original report of Cohen,

Marinello and Back (22) was widely publicized by the mass media. As a result, the significance of their findings was considerably over-emphasized, and many premature conclusions were drawn for which there was not sufficient scientific justification.

Several important facts have to be taken into consideration before we can draw any substantial conclusions from the findings of increased chromosome breakage associated with LSD in the in vitro experiments. It must be emphasized that the findings themselves were not completely consistent. In several studies there were no indications of increased chromosome breakage following the exposure to LSD. (27, 73, 105). In addition, the concentrations of LSD and durations of exposure used in these studies were usually much greater than those occurring in the human organism after the ingestion of LSD in the commonly used dosages. Cohen, Marinello and Back (22) themselves did not find increased breakage of chromosomes at the lowest concentration and time (0.001 micrograms of LSD per cc for four hours). Loughman et al. (70) emphasized that it is precisely the lowest concentration and duration of exposure used in this study that most closely approximates the expected concentration in blood, liver and other organs after a dose of 100 micrograms of LSD ingested by a man weighing 70 kg. If the metabolic degradation of LSD is considered, then the effective concentration in vivo of unchanged LSD would be considerably less than this, approximating 0.0001 micrograms per cc—a concentration used only by Kato and Jarvik, (65) who found no increase in breakage at this dosage.

In general, special caution is required in extrapolating the in vitro findings to the situation in the living organism. The intact human organism differs fropi isolated cells in the test tube in its enormous complexity and in its ability to detoxify and excrete noxious compounds. Substances that are toxic in vitro do not necessarily have the same effect in vivo. In addition, some of the techniques used in the in vitro studies can create an artificial situation and introduce factors that do not exist in the living organism. This issue has been discussed in detail in an excellent review on LSD and genetic damage by Dishotsky et al. (28) These authors point to the fact that all the studies on cultured lymphocytes have used modifications of a technique in which the lymphocytes are stimulated by phytohemagglutinin to enter the reproductive cell cycle. In the normal state in vivo, small lymphocytes are in a phase of growth which precedes DNA synthesis; they do not grow, divide or enter the cell cycle. Thus, in the studies in vitro, lymphocytes are exposed to chemical agents during developmental stages of the cell cycle, including the synthesis of DNA, which do not normally occur in these cells in the body. Damage to a lymphocyte in this phase generally will not manifest itself as chromatid-type change in a subsequent division. Most, if not all chromatid-type changes are initiated by technical procedures, and the great majority of lesions reported in the in vitro and in vivo studies were of the chromatid type. The findings of an increased rate of chromosomal breakage in lymphocytes exposed to LSD in vitro must therefore be interpreted with great caution.

Many recent studies concerning the structural changes caused in chromosomes by LSD gave the impression that this effect was something specific and unique. Most of these reports have silently bypassed a fact that would have made the issue much less interesting and sensational. The changes in chromosomal structure described are not exclusively caused by LSD; they can be induced by a variety of other conditions and substances. Factors that have been known to cause chromosomal breakage in vitro include radiation, changes in temperature, variations in oxygen pressure, impurities in tap water unless it is distilled twice, and a variety of common viruses. The long list of chemical substances that increase the chromosomal breakage rates contains many commonly used drugs, including aspirin and other salicylates, artificial sweeteners, the insecticide DDT, morphine, caffeine, theobromine, theophylline, tranquillizers of the phenothiazine type, sonic vitamins and hormones, and many antibiotics such as aurcomycin, Chloromycetin, terramycin, streptomycin and penicillin.

In this connection it is interesting to quote Sharma and Sharma, (91) who have written an extensive summary of the literature on chemically induced chromosome breaks: "Since the first induction of chromosomal mutations by chemicals and the demonstration of definite chromosome breakage by Oehlkers, such a vast multitude of chemicals have been shown to possess chromosome breaking properties that the problem has become increasingly complex." Jarvik, (61) discussing the paper by Judd, Brandkamp and McGlothlin, (64) was even more explicit: ". . and it is likely that any compound added at the appropriate time, in the appropriate amount, to the appropriate cell type, will cause chromosome breaks."

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