Mechanisms and chemical induction of aneuploidy in rodent germ cells

The objective of this review is to suggest that the advances being made in our understanding of the molecular events surrounding chromosome segregation in non-mammalian and somatic cell models be considered when designing experiments for studying aneuploidy in mammalian germ cells. Accurate chromosome segregation requires the temporal control and unique interactions among a vast array of proteins and cellular organelles. Abnormal function and temporal disarray among these, and others to be identified, biochemical reactions and cellular organelles have the potential for predisposing cells to aneuploidy. Although numerous studies have demonstrated that certain chemicals (mainly those that alter microtubule function) can induce aneuploidy in mammalian germ cells, it seems relevant to point out that such data can be influenced by gender, meiotic stage, and time of cell-fixation post-treatment. Additionally, a consensus has not been reached regarding which of several germ cell aneuploidy assays most accurately reflects the human condition. More recent studies have shown that certain kinase, phosphatase, proteasome, and topoisomerase inhibitors can also induce aneuploidy in rodent germ cells. We suggest that molecular approaches be prudently incorporated into mammalian germ cell aneuploidy research in order to eventually understand the causes and mechanisms of human aneuploidy. Such an enormous undertaking would benefit from collaboration among scientists representing several disciplines.     

Lack of correlation between mutagen-induced chromosomal univalency and aneuploidy in mouse spermatocytes

Aneuploidy represents the predominant type of chromosomal abnormality found in human newborns with birth defects. The concern that environmental agents may cause aneuploidy in germ cells has prompted development of assay systems for detection of potentially aneuploidy-producing agents. One of the most frequently used methods involves cytogenetic analysis of murine spermatogenic cells at the stages of meiotic metaphases. However, criteria for aneuploidy induction have not been standardized in this test system. Many investigators consider the ability of an agent to induce univalents an appropriate measure of its potential to induce aneuploidy. In the present study, the relationship between univalency and aneuploidy was examined in mouse spermatocytes after various mutagen treatments. 45 Swiss mice were treated with 4 different agents; viz., adriamycin vinblastine sulfate, cytosine arabinoside, and radiation (cobalt 60) and 10 untreated animals served as controls. From each animal, 50–200 MIs were examined for both sex-chromosomal and autosomal univalency (X-Y U and AU), and equal numbers of MIIs were examined for aneuploidy (hyperhaploidy). No significant correlations between univalency (either X-Y U or AU) and aneuploidy were found in the mutagen-treated mice; nor were they found in the untreated animals. These results indicate that induction of univalents by a mutagen is not necessarily predictive of the aneuploidy-inducing ability of his agent.     

Germ-cell nondisjunction in testes biopsies of men with idiopathic infertility.

Intracytoplasmic sperm injection (ICSI) has been used in combination with testicular sperm extraction to achieve pregnancies in couples with severe male-factor infertility, yet many of the underlying genetic mechanisms remain largely unknown. To investigate nondisjunction in mitotic and meiotic germ cells, we performed three-color FISH to detect numeric chromosome aberrations in testicular tissue samples from infertile men confirmed to have impaired spermatogenesis of unknown cause. FISH was employed to determine the rate of sex-chromosome aneuploidy in germ cells. Nuclei were distinguished as haploid or diploid, respectively. The overall incidence of sex-chromosome aneuploidy in germ cells was found to be significantly higher (P<.00001) in all three abnormal histopathologic patterns (range 39.0%-43.5%) as compared with normal controls (29.1%). The relative ratio of normal to aneuploid nuclei in the diploid cells of patients with impaired spermatogenesis was approximately 1.0, a >300% decrease when compared with the 4.42 ratio detected in patients with normal spermatogenesis. These results provide direct evidence of an increased incidence of sex-chromosome aneuploidy observed in germ cells of men with severely impaired spermatogenesis who might be candidates for ICSI with sperm obtained directly from the testis. The incidence of aneuploidy was significantly greater among the diploid nuclei, which suggests that chromosome instability is a result of altered genetic control during mitotic cell division and proliferation during spermatogenesis.     

A genetic assay for the detection of aneuploidy in the germ-line cells of Drosophila melanogaster

A 2-generation assay is described for the detection of aneuploidy in the germ-line cells of Drosophila melanogaster. Larvae and adult females that carry marker mutations are exposed to test compounds, and the F2 generation is scored for exceptional phenotypes. As a consequence of nondisjunction and/or loss of the sex chromosomes, 5 exceptional phenotypes appear. These phenotypes are often indicative of specific types of nondisjunction. Based on the time course and the pattern of exception production of the treated parents, aneuploidy due to meiotic and mitotic defects can be separated. The genetic analysis of the exceptions reveals whether nondisjunction has occurred due to failure of the spindle fibres to disjoin chromosomes or attachment of the chromosomes. The described assay is an extension of the so-called Somatic Mutation and Recombination Test (SMART) and allows screening for different genetic endpoints: aneuploidy, recombinogenic and mutagenic activities in the same treatment. The effects of colchicine and EMS are described with respect to the induction of aneuploidy in the germ line and somatic mutation and recombination in the eyes, wings and female germ-line cells. Colchicine induces aneuploidy in the germ-line cells while the frequency of mosaic spots does not increase after colchicine treatment. This result suggests that aneuploidy plays little (if any) role in the formation of mosaic spots. Colchicine induces nondisjunction in the mitotically rather than in the meiotically dividing germ-line cells. EMS, as expected, induces high frequency of somatic mutation and recombination but not aneuploidy in the female germ line.     

Mammalian in vivo assays for aneuploidy in female germ cells

This paper presents an evaluation of and offers recommendations for assays to detect chemically induced aneuploidy in mammalian female germ cells. 72 papers on female germ cell aneuploidy, published from 1970 to 1984, were reviewed. 28 papers were selected for critical evaluation; the other 44 papers were rejected according to pre-established criteria. Salient points emerging from the information reviewed allow an assessment of the current status of mammalian female germ cell tests for aneuploidy. The majority of data have been obtained by analyzing metaphase II mouse oocyte chromosomes following superovulation. Various classes of chemicals were administered usually around the time of ovulation. Dose-response relationships have not been obtained for the majority of chemicals evaluated. The method of data reporting and analysis usually was not conducive to comparisons among different studies. Few of the 16 chemicals studied can be regarded as negative for their ability to induce aneuploidy, whereas an even smaller number should be considered as positive. Certainly, a need exists to identify the chemicals and the dosages that could increase the incidence of aneuploidy in mammalian female germ cells. Obtaining such data definitely is feasible in cytogenetic laboratories. However, the mammalian female germ cell aneuploid assay should not be perceived as a rapid, inexpensive, routine procedure. The assay is capable of detecting aneuploidy following anaphase I when metaphase II oocytes are studied and following anaphases I and II when first-cleavage zygotes are studied.     

Persistence of aneuploid immature/primitive hemopoietic sub-populations in mice 8 months after benzene exposure in vivo

Benzene (bz) is a common environmental contaminant associated with increased risk of myeloid leukemia. Chronic bz exposure in vivo increases the frequency of aneuploid circulating lymphocytes in humans. However, there is no information about persistence of bz-associated aneuploidy in immature/primitive cells, at risk of leukemic transformation, after bz exposure in vivo. We explored the relationship between the induction and persistence of aneuploidy in primitive hemopoietic cells from mice that received oral doses of bz in vivo. Short- and long-term persistence of aneuploidy were evaluated in immature/primitive sub-populations (Lin(-)c-kit(+)Sca-1(+)), as well as lymphoid and myeloid cells, 6 days and 2-8 months after exposure. Mice receiving bz in a corn oil carrier, or corn oil alone, both have increased aneuploidy frequencies (1-5%, compared to <1% in untreated controls) in all sub-populations, 6 days after exposure. However, unlike bz-induced aneuploidy, corn oil-induced aneusomies are transient, with frequencies returning to background levels in lymphoid and myeloid cells, 9 weeks after exposure. The frequency (5-9%) of aneuploid lymphocytes and myeloid cells is higher at 9 weeks than at 6 days, suggesting that bz disrupts chromosomal segregation in differentiated cells and/or progenitors. About 8 months after bz exposure, the Lin(-)c-kit(+)Sca-1(+) sub-population contains up to 14% aneuploid cells with numerical chromosomal aberrations affecting chromosomes 2 or 11. These data demonstrate that bz induces DNA copy number changes in immature/primitive cells, and that these changes persist for long periods. Although, initial exposures are not leukemogenic, subsequent exposures of cells to genotoxins or oxidative radicals that induce additional genetic hits may increase the risk of transformation. The contribution of bz-induced aneuploidy in immature/primitive cells to leukemogenesis remains to be determined.     

Generation of a novel isogenic trisomy panel in human embryonic stem cells via microcell-mediated chromosome transfer

Aneuploidy is the gain or loss of a chromosome. Down syndrome or trisomy (Ts) 21 is the most frequent live-born aneuploidy syndrome in humans and extensively studied using model mice. However, there is no available model mouse for other congenital Ts syndromes, possibly because of the lethality of Ts in vivo, resulting in the lack of studies to identify the responsible gene(s) for aneuploid syndromes. Although induced pluripotent stem cells derived from patients are useful to analyse aneuploidy syndromes, there are concerns about differences in the genetic background for comparative studies and clonal variations. Therefore, a model cell line panel with the same genetic background has been strongly desired for sophisticated comparative analyses. In this study, we established isogenic human embryonic stem (hES) cells of Ts8, Ts13, and Ts18 in addition to previously established Ts21 by transferring each single chromosome into parental hES cells via microcell-mediated chromosome transfer. Genes on each trisomic chromosome were globally overexpressed in each established cell line, and all Ts cell lines differentiated into all three embryonic germ layers. This cell line panel is expected to be a useful resource to elucidate molecular and epigenetic mechanisms of genetic imbalance and determine how aneuploidy is involved in various abnormal phenotypes including tumourigenesis and impaired neurogenesis.     

In vitro and in vivo extrapolations of genotoxin exposures: consideration of factors which influence dose-response thresholds

The concept of a threshold of activity of a genotoxic agent is primarily based upon considerations of protective mechanisms and multiple cellular targets, which require inactivation before a toxic response is produced. In this paper, we have considered and evaluated the influences of compound metabolism, DNA lesion formation, mutation induction and sequence content, aneuploidy induction and the influence of repair enzymes upon genetic endpoints produced by both DNA reactive chemicals and by those chemicals which modify non-DNA cellular targets. Thresholds of activity have been evaluated by critical analysis of the published literature and original data analysing both the role of sequence context upon point mutation induction and DNA repair mechanisms upon the sensitivity of cultured cells to the induction of aneuploidy. In the case of DNA reactive chemicals, the presence of a threshold of chemical activity will be dependent upon cellular activities such as those of the Phase II enzymes reducing the activity of chemicals before lesion formation takes place and/or those of the DNA repair enzymes which reduce the proportion of DNA lesions which are processed into DNA sequence changes. Under such conditions, a given exposure of a DNA reactive chemical does not produce a linear or semi-linear increase in DNA lesions or in mutation frequency. However, even when these protective mechanisms are overwhelmed by the high exposures of genotoxic chemicals the biological effects of a genotoxin may be influenced by the sequence context of the gene under consideration. Here, we demonstrate that point mutations are detected at relatively higher frequencies in the non-coding introns compared with the coding exons. Many of the base changes detected in the exons do not produce amino acid changes in the proteins coded for by the genes being monitored for mutation induction. Both sequence context and the types of base changes induced may provide a "buffering" effect reducing the biological consequences of mutation induction. Spindle damaging chemicals, such as colcemid and vinblastine, induce aneuploidy by modifying the numbers of spindle fibres which regulate the segregation of chromosomes during mitosis and meiosis. The redundancy of spindle fibres in the dividing mammalian cell leads to the prediction that only chemical exposures which damage most, if not all, of the fibres will lead to the induction of polyploidy and/or aneuploidy. Such predicted thresholds of chemical activity can be observed when both chromosome loss and non-disjunction are measured in wild type cultures. However, we observed a substantial increase in sensitivity to aneugenic chemicals when measurements were made in primary cell cultures derived from xerodoma pigmentosum and trichothiodystrophy patients. Further studies are necessary to evaluate the consequences of the genetic background of tester strains upon the nature of the dose-response curve of aneugenic chemicals.     

Telomerase abrogates aneuploidy‐induced telomere replication stress,senescence and cell depletion

The causal role of aneuploidy in cancer initiation remains under debate since mutations of euploidy‐controlling genes reduce cell fitness but aneuploidy strongly associates with human cancers. Telomerase activation allows immortal growth by stabilizing telomere length, but its role in aneuploidy survival has not been characterized. Here, we analyze the response of primary human cells and murine hematopoietic stem cells (HSCs) to aneuploidy induction and the role of telomeres and the telomerase in this process. The study shows that aneuploidy induces replication stress at telomeres leading to telomeric DNA damage and p53 activation. This results in p53/Rb‐dependent, premature senescence of human fibroblast, and in the depletion of hematopoietic cells in telomerase‐deficient mice. Endogenous telomerase expression in HSCs and enforced expression of telomerase in human fibroblasts are sufficient to abrogate aneuploidy‐induced replication stress at telomeres and the consequent induction of premature senescence and hematopoietic cell depletion. Together, these results identify telomerase as an aneuploidy survival factor in mammalian cells based on its capacity to alleviate telomere replication stress in response to aneuploidy induction.     

植物非整倍体研究进展

非整倍体(aneuploid)是指相对于正常个体(euploid)的染色体组增加、减少一条或若干条染色体的生物个体。由于非整倍体个体存在基因剂量效应的不平衡性(gene-dosage imbalance),非整倍体个体往往会表现严重的表型缺陷(aneuploid syndrom),如发育迟缓,个体矮小,难以繁殖后代等。在人类中,最为典型的例子为导致新生儿智力缺陷的唐氏综合症,由额外的一个21号染色体拷贝(部分拷贝)引起。此外,大多数癌细胞类型表型为严重的非整倍体。在大多情况下,非整倍体对于动物及人类是致命的,而植物对于非整倍体则往往表现出较强的耐受力,特别是在异源多倍体植物中。植物非整倍体对于植物的遗传、育种研究有重要意义,在基因及分子标记的物理位置确定,基因转移,连锁群与染色体的对应关系的确立上具有无可比拟的优势。该文综述了近些年来有关植物非整倍体研究的结果,介绍了非整倍体的几种重要成因和有关非整倍体鉴定手段的变迁,阐述了植物非整倍体对个体表型、基因表达以及表观遗传方面的影响,重点讨论了非整倍体在植物进化、基因组序列测定以及遗传改良方面的潜在作用。同时,探讨了植物非整倍体研究的新思路,以及利用非整倍体促进相关植物遗传改良、育种研究的新方法。     

Chemically-induced aneuploidy in mammalian oocytes

The ability of certain chemicals to elevate the frequency of aneuploidy above spontaneous levels in mammalian experimental models prompts the concern that a similar situation might exist in humans. Validation of experimental models for aneuploidy studies is in progress since there is much to be learned about the causes and mechanisms of chemically-induced aneuploidy. Several biological variables have been shown to influence the results from aneuploidy assays. In this review, we examine these variables as they relate to female germ cell aneuploid assays. Also, we have found that the aneuploidy results obtained from different cell types, sexes, and experimental models cannot necessarily be expected to agree due to certain anatomic and physiologic differences and the end points measured.     

hCDC4 and Genetic Instability in Cancer

About twenty years ago, scientists began to discover that colorectal cancers are caused by the sequential acquisition of genetic alterations in specific genes. To this day, we are still dissecting the genome of colorectal cancers to identify specific “culprit” genes that play a role in tumorigenesis. At the same time, we have more recently begun to turn our attention to the features of cancer cells that distinguish them from normal cells and that may be targeted therapeutically. Aneuploidy is one such hallmark of cancers, but its role in tumorigenesis is heretofore undetermined. Our efforts have focused on elucidating the fundamental mechanisms underlying aneuploidy. The assertion that a genetic basis for aneuploidy would imply its importance in tumorigenesis, and consequently make it a potential therapeutic target, represents the rationale for our pursuit of this line of research. For the last few years, we have been trying to determine whether there is a genetic cause underlying this attribute of cancers. Our recently published work entitled, “Inactivation of hCDC4 can cause chromosomal instability,” attempts to address this issue and raises more questions about the cause, mechanism, timing, and therapeutic potential of genetic instability.1     

Chromosomal abnormalities and the morphology of mouse sperm heads.

In the mouse, numerous mutagens, teratogens and carcinogens have been shown to induce marked elevations in the fraction of sperm with head shape abnormalities. Since carcinogens and teratogens may act by causing genetic damage, a likely explanation of these results is that the sperm abnormalities are also caused by genetic damage. There are two more or less distinct classes of genetic damage, chromosomal aberrations and point mutations. In this paper, we provide evidence, that in general, chromosomal aberrations are not responsible for causing abnormally shaped sperm. Chromosomal aberrations could have caused abnormal sperm morphology in a number of ways. One possibility was that the mere presence of a translocated chromosome within the germ cell led to the malformation of the sperm head. A second possibility was that chromosomal imbalance, i.e., aneuploidy, duplications or deficiencies, within the spermatid or haploid cells caused abnormalities in shape. We tested these hypotheses by measuring the level of abnormally shaped sperm in mice homozygous and heterozygous for 24 various reciprocal and Robertsonian translocations. The diploid cells of these mice are known to be chromosomally balanced, containing translocated chromosomes. A predictable proportion of their gametes are, however, chromosomally unbalanced and carry translocated chromosomes. It was found that the levels of sperm abnormalities in these mice were convincingly unrelated to the levels predicted by any of the above hypotheses. Based on these results it seems that sperm abnormalities in mice are not due to the mere presence of translocated chromosomes in germ cells and also not due to chromosomal aneuploidy or duplication-deficiencies of chromosomal segments in the spermatid during development of the sperm.     

Review of literature on chemical-induced aneuploidy in mammalian male germ cells

80 papers published between 1970 and 1984 were evaluated for results pertaining to chemical-induced aneuploidy in mammalian male germ cells. Diverse assays and end points were represented. The assays considered to involve direct measures of aneuploidy were based upon chromosome counts in premeiotic, meiotic, and embryonic cells, and the male pronucleus, or upon phenotypic expression of X-linked genetic markers. Assays in which indirect measures were interpreted as evidence for aneuploidy included those primarily assessing chiasma frequencies, univalent frequencies, and spermatid/sperm sex chromosome body counts. An initial screening to reject studies with insufficient data and those which did not involve a single chemical test agent led to the elimination of 39 papers from further review. The remaining 41 papers reported effects from 46 different chemicals. These papers were rigorously assessed for adequacy of experimental protocols, relevance of end points as direct measures of aneuploidy, and completeness of data presentation and statistical analysis. Criteria specific to each assay were also considered. 4 chemical tests were considered to provide reliable positive or negative aneuploidy data. Cyclophosphamide and chloral hydrate each caused metaphase II hyperploidy when injected into mice. Very limited analyses of trenimon and isoniazid provided negative results. Test findings for 44 chemicals were viewed as inconclusive. It was concluded that standardization of tests to evaluate chemical-induced aneuploidy in male germ cells and the application of these tests towards increasing the data base are badly needed.     

The response of germ cells of the mouse to the induction of non-disjunction by X-rays

The sensitivity of male and female pre-meiotic germ cells of the mouse to the induction of non-disjunction by low doses of X-rays, has been tested. No enhancement with 5 rad was observed over control of values in dictyate oocytes irradiated from young or aged females. In males, a 3-fold increase in overall chromosome abnormalities (aneuploids, polyploids and mosaics) was found following the treatment of germ cells sampled in the 7th week after irradiation (spermatogonia and early primary spermatocytes) with 100 rad. The increase in aneuploidy alone was not however significant at the 5% level of probability. Primary spermatocytes sampled in week 5 after irradiation were generally insensitive to the induction of chromosome abnormalities.     

Aging and diethylstilbestrol-induced aneuploidy in male germ cells: a transgenic mouse model

The incidence of aneuploidy in male germ cells was evaluated by analyzing extra marker chromosome(s) signal(s) in round and/or hook spermatids of transgenic mice. Two types of transgenic mice were used as models. The inserted foreign DNA (λ-gt10LacZ shuttle vector and/or pSVc-myc plasmid) was located at the middle of the long arms of chromosome 2 (λ DNA) and/or chromosome 8 (c-myc). The number of marker chromosomes present could easily be detected after fluorescence in situ hybridization (FISH) in testicular cells. The frequency of spontaneous aneuploidy of chromosome 2 was similar in round spermatids of lambda and λ-myc mice. Differential involvement of chromosomes 2 and 8 was observed in both round and hook spermatids. The frequency of spontaneous aneuploidy in round spermatids was higher than that in hook spermatids. The frequency of aneuploidy of marker chromosomes was significantly higher in older mice (2 years old) than in younger ones. Diethylstilbestrol (DES)-induced aneuploidy was dose dependent, and was not influenced by the stage at which germ cells were treated with DES. These results demonstrate the usefulness of a transgenic mouse model for the study of aneuploidy in germ cells. Received: 5 August 1998 / Accepted: 27 August 1998     

In vitro gametogenesis from embryonic stem cells

Many insights into mammalian germ cell development have been gained through genetic engineering and in vivo studies, but the lack of an in vitro system for deriving germ cells has hindered potential advances in germ cell biology. Recent studies have demonstrated embryonic stem cell differentiation into germ cells and more mature gametes, although significant unanswered questions remain about the functionality of these cells. The derivation of germ cells from embryonic stem cells in vitro provides an invaluable assay both for the genetic dissection of germ cell development and for epigenetic reprogramming, and may one day facilitate nuclear transfer technology and infertility treatments.     

Cell adaptation to aneuploidy by the environmental stress response dampens induction of the cytosolic unfolded-protein response

Aneuploid yeast cells are in a chronic state of proteotoxicity, yet do not constitutively induce the cytosolic unfolded protein response, or heat shock response (HSR) by heat shock factor 1 (Hsf1). Here, we demonstrate that an active environmental stress response (ESR), a hallmark of aneuploidy across different models, suppresses Hsf1 induction in models of single-chromosome gain. Furthermore, engineered activation of the ESR in the absence of stress was sufficient to suppress Hsf1 activation in euploid cells by subsequent heat shock while increasing thermotolerance and blocking formation of heat-induced protein aggregates. Suppression of the ESR in aneuploid cells resulted in longer cell doubling times and decreased viability in the presence of additional proteotoxicity. Last, we show that in euploids, Hsf1 induction by heat shock is curbed by the ESR. Strikingly, we found a similar relationship between the ESR and the HSR using an inducible model of aneuploidy. Our work explains a long-standing paradox in the field and provides new insights into conserved mechanisms of proteostasis with potential relevance to cancers associated with aneuploidy.     

The TX;Y test for the detection of nondisjunction and chromosome breakage in Drosophila melanogaster. II. Results of female exposures

The TX; Y test is a short-term assay for the detection of sex-chromosome nondisjunction and chromosome breakage in Drosophila melanogaster. It has been used in previous work following the exposure of males. In this work, females are exposed. When females are the exposed parent, only chromosome gain can be detected. Positive results for the induction of aneuploidy were obtained following exposures of females to X-rays, 10 degrees C cold shock, and colchicine. No increase in aneuploidy was obtained following exposures of females to DMSO and trifluralin. Comparison with similar work in males reveals no consistent pattern concerning the more appropriate sex to use for aneuploidy testing in Drosophila, as colchicine was found to be positive in females only and DMSO and trifluralin were effective in males only. Further work is necessary to validate the TX; Y test and to understand the relative efficacy of female and male exposures to aneuploidy inducing agents in Drosophila.     

Current status of aneuploidy testing in Drosophila

Based on the literature on file at EMIC, 181 papers contained material on aneuploidy testing. Initial screening rejected papers providing no data, no negative control and/or poorly designed genetic schemes; 67 papers representing tests of 76 compounds were reported on. Statistical classifications were established as follows: (+)=a statistically significant difference at the 5% level between the treated and control frequencies; (-)=no significant difference at the 5% level when the number of offspring tested was sufficient to have identified an increase of 0.2% over the control with a power of 75%; I=inconclusive= (a) no significant difference at the 5% level but the number of offspring tested was below that necessary to detect an increase of 0.2% with a power of 75%; (b) the nature of apparent complete loss is undetermined; or (c) the nature of the germ cells sampled is not determined. Of the 76 compounds analyzed, calls were made on 34 compounds. 17/34 compounds were positive for chromosome gain (11/34 for chromosome gain and chromosome loss, 6/34 for chromosome gain only). 17/34 compounds were negative for chromosome gain (11/34 for chromosome gain and chromosome loss and 6 for chromosome gain only). Are any fo the compounds found to induce aneuploidy specific for aneuploid induction? 7 or the compounds positive for chromosome gain were positive in one or more tests assaying for other genetic endpoints, and no reliable data exists regarding results in other tests for the remaining 10 compounds; accordingly, the answer to the question awaits further work.