In Vivo Studies

Because of the limitations of the in vitro approach, in vivo studies are preferred for assessing the possible genetic dangers associated with administration of LSD. Unfortunately, of the twenty-one reports that have been published by seventeen laboratories many have serious methodological shortcomings and are more or less inadequate, while individual reports contradict each other and their overall results are inconclusive. Two major approaches have been used in the in vivo studies. In fourteen of these projects, subjects were exposed to illicit substances of unknown composition and potency, some of which were alleged to be LSD. In eleven studies, individuals were exposed to known quantities of pharmaceutical^ pure LSD in experimental or therapeutic settings.

Dishotsky et al (28) published a review in which they presented a synopsis of the studies of this kind conducted prior to 1971. According to this review, of a total of 310 subjects studied, only 126 were treated with pure LSD; the other 184 subjects were exposed to illicit or "alleged" LSD. Eighteen of the 126 subjects (14.29 percent) in the group given pure LSD showed a higher frequency of chromosome aberration than the controls. In contrast, 89 of the 184 subjects (48.9 percent) in the group taking illicit LSD showed an increased incidence of aberrations—more than three times the frequence reported for subjects given pharmacologically pure LSD. Only 16.67 percent (18 of 108) of all the subjects reported to have chromosome damage, were given pure LSD. There is, therefore, good reason to discuss the two categories of in vivo studies, those with pure and those with "alleged" LSD, separately.

Illicit LSD and Chromosomal Damage

The initial findings of chromosomal damage in illicit LSD users were reported by Irwin and Egozcue. (57) They compared a group of eight illicit LSD users with a group of nine controls. The users had a mean breakage rate of 23.4 percent, more than double the 11.0 percent rate In the controls. Only two of the eight users did not have increased breakage rates. In a later and more extensive study carried out by Egozcue, Irwin and Marufjo, (33) the mean breakage rate in forty-six illicit LSD users was 18.76 percent (with a range between 8 and 45 percent); this was more than double the rate of 9.03 percent found in control cells. Only three of the forty-six users did not have a breakage rate higher than the mean control rate. In addition, the authors studied the chromosomes of four infants exposed to LSD in utero. All four showed breakage rales above the mean control value. There was no evidence of disease or physical malformation in any of these children.

These findings were supported by Cohen, Hirschhorn and Frosch, (20) who studied eighteen subjects exposed to illicit LSD. They described an increased chromosomal breakage in this group (mean 13.2 percent) which was more than triple that of the control group (3.8 percent). The authors also examined the chromosomes of four children born to three mothers who took LSD during pregnancy. The frequency of chromosome breaks was elevated in all four, and was greater in the two children who were exposed to LSD during the third and fourth months of pregnancy than in the two infants exposed to low doses of LSD late in pregnancy.

In a later paper, Cohen et al. (21) reported that thirteen adults exposed to illicit LSD showed chromosome breakage rates that were above the control mean. In nine children exposed to illicit LSD in utero, they found a mean breakage of 9.2 percent, as compared with 4.0 percent in four children whose mothers had used illicit LSD before but not during pregnancy. The breakage rate in the control group was 1.0 percent. All but two children had been exposed to other drugs during pregnancy; all were in good health and showed no birth defects.

Nielsen, Friedrich and Tsuboi (82) found that their ten subjects exposed to illicit LSD had a mean breakage rate of 2.5 percent; this was significantly higher than that of the control group (0.2 percent). However, the allegedly pathological 2.5 percent rate is lower than that of the controls in other positive studies.

A number of investigators have not been able to demonstrate increased chromosome breakage in LSD users. The synoptic paper by Dishotsky et al. (28), quotes nine groups of researchers who reported negative results of similar studies. At the present time, therefore, the results of the in vivo studies are considered rather controversial and at best inconclusive.

Many investigators have attempted to offer explanations for the existing discrepancies between positive and negative reports. Some have criticized the breakage rate for controls in the studies by Cohen et al. (21) (3.8 percent) and Irwin and Egozcue (57) (11.9 percent and 9.03 percent) as being unusually high. Others have suggested that the high control values could have resulted from viral contamination of the cultures, insufficiently fortified media interfering with chromosome repair, technical variation in cell culturing, and the approach to chromosome evaluation. It was also pointed out that in these studies, chromosome-type and chromatid-type changes were not reported separately but were combined and then converted to "equivalent numbers of breaks." Combining the two types of aberrations in a single index obscures the distinction between real chromosome damage occurring in vivo and damage arising in the course of cell culture.

However, these factors cannot account for the discrepancies between the findings of various teams of investigators. If they did, the aberrations resulting from these effects would be randomly distributed between groups exposed to illicit LSD and control groups. Since the distribution is uneven, these factors do not explain the significantly elevated breakage rates in eighty of the eighty-six subjects exposed to illicit LSD studied by Cohen et al. and by Irwin and Egozcue.

A much more important clue to the understanding of this controversy seems to be related to certain characteristics of the group of the "LSD users." In this type of research, the investigators depend on the recall and reliability of the subjects in determining the type of drugs they have used in the past, the number and frequency of exposures, the alleged dosages, and interval since last exposure. Even in cases where the reports are accurate, the subjects usually do not know the content and the quality of the samples they are using. The content of pure LSD in the illicit LSD samples is almost always questionable, and various impurities and admixtures rather frequent. The samples analyzed in the past have been demonstrated to contain amphetamines, mescaline, DOM (4-methyl-2, 5-dimethoxyam-phetamine, also called STP), phencyclidine (phenylcyclohexylpiperidine, PCP or "angel dust"), benactyzine and even strychnine. In addition, all the subjects tested used or abused drugs other than street LSD. These drugs included, among others, Ritaline, phenothiazines, alcohol, amphetamines, cocaine, barbiturates, heroin and other opiates, and various psychedelic substances such as marihuana, hashish, psilocybin, mescaline, STP, methylenedioxyainphetamine (MDA), and dimethyl-tryptamine (DMT). Under the circumstances, one questions the logic of referring to this group in scientific papers as "LSD users." Most of these subjects were actually multiple-drug users or abusers exposed to a variety of chemicals of unknown composition, quality and potency.

In addition, it has been repeatedly reported that this population suffered from malnutrition and had very high rates of venereal disease, hepatitis and various other viral infections. It was mentioned above that viruses are one of the most common factors causing chromosomal damage; the possible role of malnutrition remains to be evaluated. Dishotsky et al. (28) conclude their review of the in vivo studies involving illicit LSD by relating the findings of increased chromosome breakage to a combination of factors such as long-term excessive exposure to illicit chemical agents, the presence of toxic contaminants, the intravenous route of administration, and the physical debility of many drug abusers. According to them, positive results, when found, are related to the more general effects of drug abuse and not, as initially reported, specifically to the use of LSD.

Pure LSD and Chromosomal Damage

Chromosomal studies of persons who received pharmaceutical^ pure LSD in an experimental or therapeutic framework are much more relevant and reliable as a source of information than the studies of illicit drug users. In these studies, there is no uncertainty concerning purity, dosage, frequency of exposure and the interval between the latest exposure and blood sampling. Two different approaches can be distinguished in the chromosome studies using pure LSD. The studies of the first type are retrospective and use a "post hoc" design; they examine the chromosomal changes in subjects who were exposed to pure LSD in the past. The studies of the second type are prospective-, the chromosomal patterns are examined both before and after the exposure to LSD, and each subject serves as his own control.

Retrospective Studies of Chromosomal Changes in Pure LSD Users. A review of the studies in this category reveals that only two groups of investigators have reported an increased rate of chromosome breakage in their subjects. Five other teams failed to confirm these positive findings.

Cohen, Marinello and Back (22) reported in their initial study that they found chromosomal damage in the white blood cells of one paranoid schizophrenic patient who had been treated fifteen times in the past with LSD in dosages between 80 and 200 micrograms. Nielsen, Friedrich and Tsuboi (80) examined the chromosomes of five persons treated with LSD and found "no correlation between any specific drug and the frcqucncy of gaps, breaks, and hyper-diploid cells." The authors later regrouped their data, forming smaller groups on the basis of age and sex. (81) After this revision of the original material, they concluded that LSD induced chromosomal damage. Tjio, Palinke and Kurland (106) criticized this study on the basis of the insufficient number of cells analyzed for a reliable determination of breakage rates. Three of the five LSD subjects studied had no chromosomal aberrations, and the two remaining subjects accounted for all six breaks found. In addition, the 1.7 percent breakage rate is still within the values reported for the general population. Another study by Nielsen, Friedrich and Tsuboi (82) which reported an increased breakage rate of 4.3 percent in a group of nine former LSD users has been criticized by Dishotsky et al. (28) on the basis of its unusual approach to data analysis.

Sparkes, Melnyk and Bozzetti (99) did not find an increase in chromosomal breakage in four patients treated with LSD in the past for medical reasons.. Negative results were also reported by Bender and Siva Sankar, (11) who examined the chromosomes of seven schizophrenic children who had been treated in the past by prolonged administration of LSD. These children received LSD daily in two divided dosages of 100 to 150 micrograms for a period of weeks or months. The frequency of chromosome breakage in this group was less than 2 percent and did not differ from that of the control group.

Siva Sankar, Rozsa and Geisler (93) studied the chromosome patterns in fifteen children with psychiatric problems who had been given LSD, UML or a combination of both. LSD was administered daily; the average dose for the whole group was 142.4 micrograms per day per patient, and the duration of therapy varied from 2 to 1,366 days. The breakage rate for the group treated with LSD was 0.8 percent, for the group treated with both LSD and UML 1.00 percent. This was not significantly higher than the rate of breakage in the controls. The patients in this study received LSD two to four years prior to the chromosome studies. The authors admitted that the effects of LSD on the leucocyte chromosomes might have been rectified over such a long period of time. In any case, this would indicate that LSD therapy has no long-lasting effects on the chromosomes.

Tjio, Pahnke and Kurland (106) published the results of chromosome analysis of a group of eight "normal" subjects who had received pure LSD in research experiments one to twenty-six times, two to fifteen months prior to giving the blood sample. The mean total chromosomal aberration rate for this group was 2.8 percent, and the individual rate in none of them exceeded the pre-LSD mean of 4.3 percent found in the patient sample.

Corey et al. (24) reported the result of a retrospective chromosomal study of sixteen patients, five of whom had been treated with LSD only, five with mescaline only, and six with LSD plus mescaline. In the eleven individuals who were clinically treated with LSD dosages ranging from 200 micrograms to 4,350 micrograms, frequency of chromosome breaks did not differ from that found in the thirteen controls. The respective frequencies were 7.8 percent for LSD, 5.6 percent for mescaline, 6.4 percent for LSD plus mescaline, and 7.0 percent for the control' group.

In an unpublished study, Dishotsky et al. examined the chromosomes of five subjects exposed in the past to pure LSD. The mean breakage rate in this group (0.40 percent) was not significantly different from that of the eight control persons (0.63 perccnt). In their review paper, Dishotsky et al. (28) indicate that fifty-eight of seventy (82.9 percent) of the subjects studied after treatment with pure LSD did not have chromosome damage. Because of incomplete data on nine of the remaining twelve subjects, they were not able to compute the precise percentage of subjects with elevated breakage rates. However, they estimated that this figure would range between 17.1 percent and 4.9 percent. All but one of the twelve subjects were reported by a single team of investigators. The authors concluded that in view of the procedures, incomplete data, questionable re-analysis of the data, and low breakage rates reported, there is no definite evidence from this type of experiment that pure LSD causes chromosome damage.

Prospective Studies of Chromosomal Changes in Pure LSD Users. The studies comparing the chromosomal changes before and after exposure to pure LSD represent the most adequate scientific approach to the problem from the methodological point of view, and are the most reliable source of scientific information. The first report in this category was published in 1968 by Hungerford et al. (55) who examined the chromosomes of three psychiatric patients before and after repeated therapeutic administrations of LSD. Blood samples were taken from all patients before any LSD therapy, one hour before and one and fourteen hours after each dose; follow-up samples were taken at intervals of one to six months. An increase in chromosome aberrations was observed after each of three intravenous injections of LSD. The increase was small in two of the three subjects; however, dicentric and multiradial figures appeared only after treatment, and acentric fragments appeared more frequently after treatment. In the follow-up study, a return to earlier levels was observed in all three patients. The data from this study indicated that pure LSD may produce transitory increases of chromosome abnormalities, but that these are no longer evident one month after administration of the final dose. The results were slightly complicated by the administration of chlorpromazine (Thorazine), which in itself can produce chromosomal aberrations. It is interesting to note that Hungerford's study is the only one in which LSD was administered intravenously.

Tjio, Pahnke and Kurland (106) reported the results of a study of thirty-two hospitalized alcoholic or neurotic patients treated with LSD in the framework of a double-blind controlled study at the Maryland Psychiatric Research Center. The dosage of LSD was 50 micrograms in eleven patients and 250-450 micrograms in twenty-one patients. The number of cells observed in this study (22,500) was more than twice the total number of cells observed in all other studies of pure LSD

users. The amount of breakage was not directly proportional to the dosage; actually those in the low-dose range showed greater increases than those on high dosage. The authors also examined a group of five persons who had taken illicit LSD from four to thirty-six times before the study. In these subjects, blood samples were drawn for seven to ten consecutive days before, during and after treatment with pure LSD either two or three times. Statistical analysis revealed no significant difference in the chromosomal aberration before and after LSD. In another prospective study, Corey et al. (24) examined the chromosomes of ten persons before and after the administration of 200-600 micrograms of pure LSD. The authors found no significant difference in the rate of chromosome breakage between the pre- and post-samples and confirmed the negative findings of the previous study.

It is interesting to mention in this connection two prospective studies of LSD-related chromosomal damage which were conducted in Rhesus monkeys (Macaca mulatto); the results of both studies were rather inconclusive. Egozcue and Irwin (32) administered high dosages of LSD (40 micrograms per kg.) four times at ten day intervals. Two of their animals showed increased chromosomal breaks, whereas the other two stayed within normal values. Kato et al. (66) described transitory changes in chromosomes after multiple, subcutaneous injections of LSD in high doses (125-1000 micrograms per kg. per injection) in Rhesus monkeys. The authors have not provided a statistical evaluation of the results; Dishotsky et al., (28) who later analyzed their data, found them statistically nonsignificant.

Dishotsky et al. (28) also offered a synoptic evaluation of the prospective LSD studies. According to them, only six of the fifty-six patients (10.7 percent) studied before and after treatment with pure LSD had elevated breakage rates; of these, three received LSD intravenously and one had a viral infection. Of these six subjects, one individual was not available for follow-up determinations; in the remaining five, breakage returned to that observed before treatment. From the total number of subjects studied before and after treatment, 89.3 percent did not have chromosome damage. The results of the prospective LSD studies are thus in agreement with the negative conclusion of five of the seven teams that studied subjects only after LSD treatment.

Chromosomal Changes in Germinal Cells

In the past, the positive findings of some chromosomal studies have been used as a basis for far-reaching speculations concerning the hereditary dangers associated with LSD. Journalists, and also several scientific workers, described their rather apocalyptic visions of the offspring of LSD users. Such speculations were rather premature, and insufficiently substantiated by experimental data. The reasoning that refers to structural abnormalities of the chromosomes as "damage" and relates them automatically to genetic hazards has serious gaps in its logic. In reality, it is not quite clear whether or not the structural changes in the chromosomes of the white blood cells have any functional significance, and whether they are associated with genetic abnormalities. There exist many chemical substances that cause chromosomal breaks but have no adverse effects on genetic mutation or fetal development. The complexity of this problem can be illustrated by the case of viruses. A variety of virus diseases (such as herpes simplex and shingles, measles, chicken pox, influenza, yellow fever, and possibly mumps) induce marked chromosomal damage without causing fetal malformations. According to Nichols, (79) one of the exceptions is rubella (German measles), a disease that is notorious for causing severe fetal malformations when acquired by the mother in the first trimester of pregnancy.

In addition to the methodological problems involved and the inconsistency of the findings discussed above, one more importunt fact has to be taken into consideration. In all the studies quoted, the effect of illicit or pure LSD, in vitro or in vivo, was assessed in the chromosomes of the white blood cells. No direct conclusions about the hereditary dangers associated with the administration of LSD can be drawn on the basis of these studies since the lymphocytes are not involved in the reproductive processes. Speculations about such dangers could be made only on the basis of chromosomal findings in germ cells such as the spermatozoids and ova, or their precursor cells. Unfortunately, the few existing studies of the chromosomes of germinal cells (the so-called meiotic chromosomes) yielded as inconclusive results as the studies of the chromosomes of somatic cells.

Skakkebaek, Phillip and Rafaelsen (95) studied meiotic chromosomes from six healthy male mice injected with large dosages of LSD (1,000 micrograms per kg); the number of injections and intervals between exposures varied. Several chromosomal breaks, gaps and unidentifiable fragments were found in the treated animals but, with a few exceptions, not in the control animals. The authors consider their finding tentative evidence that high doses of LSD may influence meiotic chromosomes in mice. They admitted that the number of abnormalities was small and technical errors could not be excluded, but concluded that the changes found could have influence on fertility, size of the litter, and the number of congenital malformations. In a later study, Skakkebaek and Beatty (94) injected four mice subcutaneously with dosages of 1,000 micrograms per kg of LSD twice a week for five weeks. Analysis carried out on a blind basis showed a high frequency of abnormalities in two of the treated mice. In addition, the spermatozoa of LSD-treated mice also showed morphological differences, with a more rounded convex side of the head and broader heads in general. The practical significance of these findings is considerably reduced by the fact that the dosages used far exceed anything used in clinical practice. A comparable dose in humans would come to 60,000-100,000 micrograms per person, which is 100 to 1,000 times more than the dosages commonly used in experimental and clinical work with LSD.

Another positive finding of meiotic chromosome damage induced by LSD was reported by Cohen and Mukherjee. (23) These authors injected thirteen male mice with a single dose of LSD at a concentration of 25 micrograms per kg. In this study the meiotic cells were apparently less vulnerable than somatic cells. However, there was an obvious tenfold increase in chromosome damage among the mice treated with LSD. This reached a maximum between two and seven days after injection, with a subsequent decrease and return to almost normal levels after three weeks, On the basis of evidence from clinical human cytogenetic studies, the authors concluded that chromosome anomalies of this type may lead to reduced fertility, congenital abnormalities and fetal wastage.

The other existing studies of the effect of LSD on meiotic cells brought essentially negative results. Egozcue and Irwin (32) studied the effects of LSD adminis tration in mice and Rhesus monkeys. The mice in this study received 5 micrograms per kg of LSD daily in a number of injections increasing from one to ten. Four adult male Rhesus macaques ingested doses of either 5, 10, 20 or 40 micrograms per kg of LSD. Six months after their single dose of LSD, three of the monkeys received four doses each, at ten-day intervals, of 40 micrograms per kg of LSD per dose. The authors reported essentially negative results in both the mice and the monkeys. In mice, occasional chromosomal breaks and fragments were observed in similar proportions in the control and the experimental groups. In the Rhesus monkeys, no significant differences were found before or after acute or chronic treatment.

Jagiello and Polani (60) published the results of a detailed and sophisticated study of the effect of LSD on mouse germ cells. They performed acute and chronic experiments on both male and female mice. The dosage of LSD in the chronic experiments ranged between 0.5-5.0 micrograms; in the acute experiments a single subcutaneous dose of 1,000 micrograms per kg of LSD was administered. The results of this study were essentially negative. The authors attributed the discrepancies with other studies to mode of administration, dosage and the animal strain involved.

In two of the existing studies, the effects of LSD on the meiotic chromosomes were tested in the banana fly, Drosophila melanogaster, an organism that has played an important role in the history of genetics. In one of these studies, Grace, Carlson and Goodman (44) injected male flies in concentrations of 1, 100 and 500 micrograms per cc. The dosage used is equivalent to approximately one liter of the same solution in humans (1,000, 100,000 and 500,000 micrograms respectively). No chromosomal breaks were observed in premeiotic, meiotic or postmeiotic sperm. The authors concluded that LSD is in a class quite distinct from that of ionizing radiation and mustard gas. If it is a mutagenic or radiomimetic agent in human chromosomes, it is not a very powerful one. In another study, Markowitz, Brosseau and Markowitz (74) fed LSD to male fruit flies in a 1 percent sucrose solution for twenty-four hours; the concentrations used were 100, 5,000, and 10,000 micrograms per cc. In these experiments, LSD had no detectable effect on chromosome breakage. The authors concluded that LSD is a relatively ineffective chromosome breaking agent in Drosophila.

Considerable caution is required in extrapolating the data about the effect of LSD on meiotic chromosomes obtained from animal experiments to humans, because of rather wide interspecies variability. The only report about the effect of LSD on human germ cells was published by Hulten et al. (54) These authors examined the testicular biopsy in a patient who had used massive doses of illicit LSD in the past, up to an alleged 1,000 micrograms. For a period of four weeks he practiced the administration of these dosages daily. There was no evidence of an increased frequency of structural chromosome aberrations in the germinal tissue of the testicles.

- Concluding this discussion of the effects of LSD on chromosomal structure, we can say that the results of the existing studies are inconclusive despite the fact that the dosages used in many experiments far exceed the doses used in clinical practice. Whether LSD causes structural changes in the chromosomes or not remains an open question. If it does, the circumstances and dosage range in which these occur have not been established, and the interpretation of these changes and their functional significance is even more problematic. This question could not be answered even on the basis of results of methodologically perfect chromosomal studies. In future research, much more emphasis should be put on the study of the effect of LSD on genetic mutation and embryonal development.

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