Thalidomide: lack of mutagenic activity across phyla and genetic endpoints.

The human and rabbit teratogen thalidomide has been tested for mutagenicity in a wide range of assays, ranging from bacterial gene mutation assays conducted in vitro to in vivo cytogenetic assays conducted using rabbits, and including a variety of human-derived tissues. Thalidomide was not mutagenic to 6 strains of Salmonella when tested both in the presence and absence of Aroclor-induced rat liver S9 mix. This inactivity was confirmed in strains TA98 and TA100 using a 1-h pre-incubation assay protocol with the same S9 mix (10% S9), and additionally, in strain TA98 using 3 concentrations of S9 (4%, 10% and 30% S9 in S9 mix). Thalidomide was not clastogenic either to cultured human lymphocytes (whole blood cultures, minus S9 mix) or to Chinese hamster ovary (CHO) cells treated in vitro. Further, no cytotoxicity was observed in purified human lymphocytes when exposed to thalidomide up to the limit of its solubility in the medium in the presence and absence of liver S9 from Aroclor-induced pregnant rabbit. The CHO assays were conducted without metabolic activation and in the presence of a variety of sources of auxiliary metabolic activation (PB/beta NP-induced rat liver S9 mix, pooled male and female human liver S9 mix, uninduced and Aroclor-induced pregnant rabbit liver S9 mix and foetal rabbit S9 mix). Thalidomide did not induce micronuclei in isolated human lymphocytes (minus S9 mix) and it was non-mutagenic to mouse lymphoma L5178Y TK+/- cells when tested to the limits of its solubility in the culture medium (+/- S9 mix). No indication of recombinogenic or clastogenic activity was observed for thalidomide when tested in Drosophila. In addition, it failed to induce chromosome aberrations in grasshopper neuroblasts when tested in the presence and absence of Aroclor-induced rat liver S9 mix. Some unusual chromosome morphologies were observed in the grasshopper cytogenetic preparations indicating a potential of thalidomide to interact with chromosomal proteins. However, this potential was not evident in the human lymphocyte micronucleus assay, and thalidomide was apparently not reactive to the proteins of the mouse skin, as it gave negative results in a mouse local lymph node assay for skin sensitizing agents. Thalidomide was inactive in bone marrow micronucleus assays conducted using males and females from two strains of mice, and female New Zealand white rabbits. It is concluded that thalidomide is neither a mutagen nor an aneugen. This conclusion is discussed within the context of the results of earlier mutagenicity studies, the recent claim that thalidomide may be a heritable germ cell mutagen to humans, and the current interest in thalidomide for the treatment of immune system-related diseases.

Maternal age effect: the enigma of Down syndrome and other trisomic conditions.

Aneuploidy is the most frequently observed chromosome abnormality in human liveborn, abortuses and oocytes. The only etiological factor that has been established is advanced maternal age for the occurrence of trisomies, particularly trisomy 21 which causes Down syndrome. The maternal age effect remains an enigma. Recent molecular data bearing on this question are reviewed as are the hypotheses that have been proposed linking nondisjunction and maternal age. Rationale is presented for a compromised microcirculation hypothesis that explains the cause of nondisjunction and why its occurrence changes with maternal age from menarche to menopause. It takes into account two facts: (1) 95% of Down syndrome children receive their extra chromosome from their mother, and in 80% or more of these the nondisjunction occurred in the first meiotic division, which is completed in the ovary. (2) The ovarian follicle containing the primary oocyte has no internal circulation. The hypothesis proposes that aneuploid oocytes arise from a concatenation of events. It begins with hormonal imbalance that causes a less-than-optimal microvasculature to develop around the maturing and mature follicles. The resulting decrease in the size of the perifollicular capillary bed reduces the volume of blood flow through the area, leading to an oxygen deficit and a concomitant increase inside the follicle of carbon dioxide and anaerobic products, such as lactic acid. This in turn causes a decrease in the intracellular pH of the oocyte that diminishes the size of the spindle, with consequent displacement and nondisjunction of a chromosome. The compromised microcirculation hypothesis explains the occurrence of aneuploidy in primary and secondary oocytes, sperm precursor cells, tumor and embryonic cells. It also explains why women of all reproductive ages may have a Down syndrome child.

A model that explains the varying frequency of aneuploid children with maternal age (J-shaped curve) as well as aneuploidy of paternal origin.

A compromised microcirculation could account for aneuploidy incidence in women of any reproductive age, the frequency varying with the probability of events leading to reduced development and/or function of the critical perifollicular capillary bed. This would explain the J-shaped curve of changing frequency of Down syndrome children with maternal age (Erickson, 1978). The seminiferous tubule of the testis, like the follicle, has no internal circulation, so small localized regions of reduced circulation could occur and result in aneuploidy. From all that we know about the deficiency of regional microcirculation in tumors (see Hall, 1978), it is reasonable to speculate that reduced pH could be responsible for some of the aneuploidy that is seen in practically all advanced tumors. As a first step in testing the model proposed here, we are beginning studies with mouse oocytes, on the assumption that ovarian conditions are responsible for the maternal age effect rather than uterine conditions (reduced rejection of trisomic fetuses). Should our model for aneuploidy induction in both germ and somatic cells prove to be correct, the molecular mechanism(s) would still have to be ascertained.

Hypothesis: some mutagens directly alter specific chromosomal proteins (DNA topoisomerase II and peripheral proteins) to produce chromosome stickiness, which causes chromosome aberrations.

Recent biochemical and molecular biological data on the composition and structure of the chromosome and the nucleus, combined with observations on the chromosomes of mutant yeast cells and grasshopper neuroblasts, offer new perspectives on mutagen-induced chromosome stickiness and its relation to chromosome breakage. A hypothesis consistent with these data states that chromosome stickiness (i) results from changes in specific non-histone proteins (topoisomerase II and the peripheral proteins) that are integral components of the chromosome and whose function is necessary for separation and segregation of chromatids, the changes being caused either by mutation in structural genes for the proteins (heritable stickiness) or by direct action of mutagens on the proteins (induced stickiness); (ii) occurs in various degrees (slight, moderate, severe, extreme) that are determined by the number of target protein molecules affected, a certain number (threshold) of affected molecules at a given site on a chromosome being required to resist the forces of anaphase movement in order to produce microscopically detectable stickiness; (iii) results from molecular events that can occur at several phases of the cell cycle (including interphase), but can only be recognized at prometaphase, metaphase and anaphase; and (iv) causes chromosome aberrations by the physical stretching and breaking of chromatids at the sticky sites; hence the breakage resulting from stickiness is a secondary effect that requires anaphase movement, in contrast to breakage resulting from direct action of mutagens on DNA.

Reactivity and fate of benzene and formaldehyde in culture medium with and without fetal calf serum; relevance to in vitro mutagenicity testing.

Gas chromatographic-mass spectrometric analyses were performed to determine the reactivity and fate of benzene (BEN) and formaldehyde (FA) in culture medium. BEN (solubility in water: approximately 500 ppm) does not react with culture medium, either with or without fetal calf serum, but its volatility, even in closed vials, is so great that 90% of a 250-ppm solution is lost to the head space after 1 h at 24 degrees C. FA, as a 37% aqueous solution, is a complex mixture that changes composition after 15-min incubation at 38 degrees C. FA is extremely reactive in culture medium containing fetal calf serum, and is much less reactive with medium components in the absence of serum. There is a dramatic increase in the number of daughter products in FA-treated medium over time, such that those seen immediately after FA is added to medium have been replaced after 60-min incubation (38 degrees C in closed vials) by many other interaction products. Methods ensuring maximum solubilization and minimal volatilization of BEN during exposure are essential for obtaining reproducible data on the mutagenic potential of BEN. The volatilization of FA from stock formalin solutions, and, more importantly, the interaction product(s) formed by this highly reactive compound with medium components, especially those in serum, are probably the critical aspects of an effective testing protocol for FA.

Formaldehyde-induced acentric chromosome fragments and chromosome stickiness in Chortophaga neuroblasts.

Embryos of the grasshopper Chortophaga viridifasciata were exposed in vitro to formaldehyde (FA), as formalin, at concentrations ranging from 10(-8)M (0.0003 ppm) to 10(-3) M (30 ppm) at 38 degrees C. A low frequency of distinct acentric chromosome fragments (0.02-0.04/cell) was observed in the neuroblasts after 1 hr exposure to 7.5 X 10(-4) or 10(-3) M FA plus 3 hr recovery, but not at lower concentrations, even with 4 hr exposure. There was no obvious relation between distinct fragment frequency and concentration of FA. Neuroblasts with sticky chromosomes were observed at 10(-4), 7.5 X 10(-4), and 10(-3) M FA, the percent of cells with slight, moderate, or severe stickiness varying with FA concentrations. Fragments were associated with the sticky chromosomes. The frequency of these sticky fragments at the two higher concentrations (0.15-0.30/cell) was greater than the frequency of distinct fragments. It is concluded that the distinct acentric fragments induced by FA result from breakage at a single sticky point (slight stickiness) between separating sister chromatids. The chromosome effects observed probably result from the action of daughter products that are formed by the interaction of FA with culture medium components, especially the fetal calf serum.

Mitomycin C effects on cell cycle progression, including inhibition of very late prophase, as seen in living neuroblasts of Chortophaga viridifasciata, with some observations on mitomycin C purity.

Observations were made on living neuroblasts (Nbs) of the grasshopper (Chortophaga viridifasciata) embryo during a 4-h recovery period following 1-h in vitro exposure to 10(-8), 10(-6), and 10(-4) M mitomycin C (MMC). None of these concentrations affected the duration of mid-mitosis (prometaphase, metaphase, anaphase), but one as low as 10(-8) M causes a small reduction in the rate at which Nbs move through the remainder of the cell cycle, primarily by retarding their progress through S. As the concentration is increased there is slower movement through S and also prophase (there are no true G1 and G2 periods in the rapidly dividing Nb: 4-h cell cycle at 38 degrees C). A significant proportion of the cells exposed to 10(-4) M are blocked for 1 or more h at very late prophase, i.e., just before nuclear membrane breakdown. In such retarded prophases the chromosomes resemble c-metaphase chromosomes even though the nuclear membrane remains intact. Mass spectrometry data revealed that one lot of the MMC used contained one or more impurities.

Chromosome fragments and other abnormalities induced by mitomycin C in the neuroblast of Chortophaga viridifasciata.

Mitomycin C (MMC) induces acentric chromosome fragments in the neuroblast (Nb) of the grasshopper embryo (Chortophaga viridifasciata) after acute and chronic exposure to concentrations ranging from 10(-8) to 10(-4) M, the dose response being essentially linear up to 10(-5) M. Because Colcemid is not used in the Nb assay, it was possible to detect two additional effects of MMC: (1) Prolonged retardation of many cells occurs when they reach very late prophase; the chromosomes continue condensing and lose their orderly prophase orientation, and the nuclear envelope becomes increasingly fragile. Such cells, which were observed after both acute and chronic exposure, give the false impression of being c-metaphases when they are fixed and squashed. The frequency of retarded very late prophases and the duration of retardation are related to MMC concentration and time of exposure. A rationale is presented supporting the idea that the events associated with retarded very late prophase result from MMC effects on the nuclear envelope. (2) MMC significantly increases the frequency of Nb's with attenuated centromeres at the beginning of early anaphase, an effect that appears to be caused by a delay in the repulsion of sister chromatids that usually occurs immediately after centromere separation begins.

Survival and PHA-stimulation of gamma-irradiated human peripheral blood T lymphocyte subpopulations.

Human peripheral blood T lymphocyte subpopulations were identified and isolated on the basis of their ability to bind IgG (T-G), IgM (T-M), or neither immunoglobulin class (T-null). Lymphocytes were exposed to 0, 0.5, 1.0, 2.5 or 5.0 Gy of 60Co gamma-rays either as a T-cell suspension or as separated T cell subsets. Survival curves, determined 5 days after irradiation, revealed that each subset has radiosensitive and radioresistant portions, and that the T-G cell is the most sensitive subset. Mitotic indices of 48-h cultures showed that the response of unirradiated T lymphocytes to PHA varied greatly among the subsets, the highest indices being obtained for the T-M and the lowest for the T-G cells. With the possible exception of the T-G cells, the subsets are relatively resistant to mitotic effects of gamma-rays. T-G cells suppress the PHA-induced mitotic response of the other T lymphocyte subsets, and this suppressor effect is radiosensitive, being abolished by 1.0 Gy. It is concluded that lymphocytes exposed to greater than or equal to 1 Gy of gamma-rays will have very few dividing B lymphocytes or T-G cells. This together with radiation-induced loss of T-G suppressor action means that the predominant lymphocyte types in mitosis after greater than or equal to 1 Gy are the radioresistant T-M and T-null cells.

The neuroblast of the grasshopper embryo as a new mutagen test system. III. Chromosome breakage induced by cyclophosphamide is greater with activation by rat hepatocytes than by S12 mix.

The promutagen cyclophosphamide (CPhos) induces chromosome breaks in neuroblasts of the 14-day-old grasshopper embryo (Chortophaga viridifasciata DeGeer, Orthoptera: Acrididae) only when activated with S12 mix or freshly isolated hepatocytes. After 1-h exposure followed by 3-h recovery, CPhos + hepatocytes (from uninduced adult rats) induces about 5 times more acentric fragments and induces them at lower doses than does CPhos + S12 mix (from phenobarbital-induced rats). Both activation systems contained equivalent amounts of microsomal protein. Hepatocytes could be used in suspension immediately after isolation, thereby obviating the delay necessitated by allowing for attachment. In the absence of CPhos, with or without activators, no chromosome aberrations were observed. Without CPhos, hepatocytes are less toxic than is S12 mix, as determined by reduction in the number of dividing neuroblasts.

Chromosome aberrations as a cause of subtle teratogenesis and use of the grasshopper neuroblast to test potential mutagens and teratogens.

The possible effects of low doses of environmental mutagens on the human embryo are discussed in terms of chromosome aberrations that could result in subtle teratogenesis, i.e., functional defects not detectable at birth. The action of a mutagen on the cells of an early stage human embryo has the potential of producing teratogenesis by inducing a viable chromosome aberration, e.g., a terminal deletion. Such an event would give rise to a mosaic individual. It is proposed that a functional defect of the central nervous system is the most likely result. The advantages of the neuroblast of the grasshopper embryo for detecting potential mutagens-teratogens are presented. In addition, the mitotic effects of mutagens which may also cause teratogenesis can be easily ascertained in great detail in the neuroblast, which has a short cell cycle (4 h at 38 degrees C) and which can be observed in the living condition.

Neuroblast of the grasshopper embryo as a new mutagen test system. II. Chromosome breakage induced by in vitro exposure of embryos to the direct-acting mutagens 4NQO, MNNG, adriamycin, and bleomycin.

The dose-response for the induction of acentric chromosome fragments was determined in neuroblasts of the grasshopper embryo (Chortophaga viridifasciata De Geer, Orthoptera: Acrididae) exposed in vitro to four direct-acting chemical known to be mutagenic, clastogenic, and carcinogenic: 4-nitroquinoline-1-oxide (4NQO), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), Adriamycin (ADM), and bleomycin (BLM). After a 1-hr exposure followed by a 3-hr recovery period (untreated cell cycle time is 4 hr), acentric fragments were observed at doses down to 1 microM 4NQO, 1.25 microM MNNG, and 0.125 microM ADM and BLM. After an 8-hr continuous exposure, acentric fragments were induced by 4NQO at a dose as low as 0.125 microM. These low concentrations also reduced the number of dividing cells. No chromosome aberrations or mitotic effects were observed in untreated embryos or in those exposed only to the solvent dimethyl sulfoxide. Because of the short cell cycle and the sensitivity of the neuroblast to the induction of acentric chromosome fragments by chemical clastogens, a minimum of time is needed to perform the test. From a comparison with the prominent clastogen test systems currently used, it is concluded that the grasshopper neuroblast test is the fastest and that it detects some agents that some systems do not. Grasshoppers have a worldwide distribution. If neuroblasts of other species prove to be as sensitive to mutagens as those of Chortophaga, investigators in many countries would have available a eukaryotic mutagen test system that is simple, fast, reproducible, and inexpensive.

Chromosome markers and evidence for clone formation in lymphocytes of a patient with Sézary syndrome.

Cytogenetic studies of 222 metaphase lymphocytes stimulated by phytohemagglutinin were carried out on a patient diagnosed clinically as having Sézary syndrome. Twenty-two cells (10%) contained 42 to 100 chromosomes. The remaining 200 cells contained 46 chromosomes and revealed evidence of clone formation; 45 were apparently normal diploid cells, but 155 were pseudodiploid with at least one long submetacentric marker in each cell. This marker was shown to have a consistent banding pattern from cell to cell. Of the 25 pseudodiploid cells karyotyped, there were other types of markers present. Normal chromosomes 2 and 17 were missing in all 25 karyotypes. There were seven set of two cells, each with an identical karyotype, suggesting subclonal formation. Many of the phytohemagglutinin-stimulated nondividing white blood cells had one or more nuclear protrusions. Cytogenetic examination of peripheral lymphocytes may be of value in diagnosing and following the course of this disease.

The relative contributions of B and T lymphocytes in the human peripheral blood mutagen test system as determined by cell survival, mitogenic stimulation, and induction of chromosome aberrations by radiation.

Ficoll-Hypaque-separated subpopulations of human peripheral blood T and B lymphocytes were exposed to 0, 0.5, 1.0, 2.5, or 5.0 Gy of gamma rays. Three parameters were examined: Survival, as measured by trypan blue dye exclusion in unstimulated cultures five days after irradiation; mitotic index, measured in phytohemagglutinin (PHA)-stimulated cultures 48 and 72 hours after irradiation; and chromosome aberration frequency, measured 48 or 60 hours after irradiation. Survival curves of T, B, and null cells are biphasic; the Do values for the radiosensitive populations of all three cell types are close to 0.6 Gy but are different for the radioresistant populations: 2.7 Gy for B cells, 4.77 Gy for T cells, and 6.03 Gy for null cells. B cells, as well as T cells, are stimulated to divide by PHA, and B cells comprise at least 10% of the mitotic figures seen in unirradiated cultures at 48 hours. The proportion of B lymphocytes in mitosis at any particular time after PHA stimulation decreases with increasing radiation dose, which reflects a higher mitotic radiosensitivity of B than of T cells. No significant difference, however, in chromosome aberration frequency was found between T and B cells.

Use of the Y-body for identification of skin source on a successfully grafted burn patient.

The use of the interphase male Y-body (fluorescent Y chromosome segment) technique with cryostat sections of both fresh and frozen-stored skin biopsies is described. A female burn patient appeared to retain her donor homografts, thereby negating the need for autografts. Since a retained homograft of this sort challenged our understanding of immunologic barriers, we applied the Y-body technique to cutaneous biopsies obtained from the patient's burn area that had been homografted with skin from a male donor, as well as control biopsies from the patient's unburned skin and normal control male and female skin. Based on clinical and cytogenetic observations, it was concluded that the most reasonable explanation for this case was that the regenerating tissue at the graft site was that of the recipient and not that of the originally grafted male skin.

Linear dose-response of acentric chromosome fragments down to 1 R of x-rays in grasshopper neuroblasts, a potential mutagen-test system.

Grasshopper-embryo neuroblasts have no spontaneous chromosome breakage; therefore they permit easy detection of agents that break chromosomes. An X-ray exposure of 1 R induces in them a detectable number of chromosome fragments. The dose-response of acentric fragment frequency fits a linear model between 0 and 128 R. Thus another cell type is added to those previously demonstrated to have no threshold dose for the induction of chromosome or gene mutations.

Preferential decrease in thymus dependent lymphocytes during storage at 4 C in anticoagulant.

Human lymphocytes stored at 4 C either as leukocyte concentrates (LCs) in citrate-phosphate-dextrose (CPD) or as whole blood anticoagulated with CPD show a rapid and marked decrease in the relative and absolute numbers of thymus derived (T) lymphocytes. Determinations were made on cells recoverable on a Ficoll-Hypaque (F-H) gradient. In evacuated LCs, the relative percentage of T cells dropped to less than 10 per cent within 72 hours with a concomitant increase in the relative percentage of bone marrow derived (B) cells to 80 per cent or more. LCs opened to the air and subsequently stored at 4 C displayed an even more precipitous decline in the relative percentage of T cells, reaching a 10 per cent level within 72 hours. The relative percentage of T cells in CPD-anticoagulated whole blood samples stored at 4 C displayed similar decreases, reaching 20 per cent levels within 24 hours. The change in the relative percentage of T cells at the Ficoll-Hypaque interface was shown to reflect a decrease in the total numbers of T cells placed on the F-H gradient with time, since determinations of T and B cell numbers in NH4Cl-treated whole blood showed a 65 to 80 per cent decrease in the numbers of T cells within 24 hours in anticoagulated whole blood held at 4 C. Thus, it may be inferred that the T cell decrease is mediated via some interaction of anticoagulant, storage time, and some component(s) present in both LCs and whole blood.