Short-term cytogenetic assays of nine cancer chemotherapeutic drugs with metabolic activation

Nine anticancer drugs were analyzed in cell cultures with respect to their cytogenetic effects with or without the addition of liver fraction S9 as an in vitro metabolic system. Among them, vincristine was the only drug which induced a significant accumulation of mitosis with or without S9. Adriamycin and neocarzinostatin were most potent in induction of chromosome breakage, but their clastogenic activities were reduced after metabolic conversion. On the other hand, cyclophosphamide had a dose-dependent activation of clastogenic effect by S9. The activity of bleomycin was enhanced by S9. S9 had no effect on the activities of actinomycin D, cytosine arabinoside, mitomycin C, and methotrexate. Vincristine showed no clastogenic property in our short-term assay system with or without S9. The activities of these anticancer drugs observed from our assay were compatible with those from other assay systems. The incorporation of S9 or other metabolic systems in routine clastogen assays should allow us to improve our understanding of the genetic toxicity of chemical agents.     

A statistical examination of historical controls for mouse bone marrow cytogenetic assays

Data from 1,111 controls from assays run over 11 years are examined to determine a most powerful statistical procedure for detecting a mutagenic effect. It is concluded that the data do not show a constant probability of chromosomal abnormalities and that the data do not fit a simple Poisson or binomial distribution. Empirical Bayes techniques are used to derive a test that declares an effect if three or more cells in a group of 50 tested contain abnormalities.     

Principles and practices of integrating genotoxicity evaluation into routine toxicology studies: A pharmaceutical industry perspective

In this article, an integrated in vivo genotoxicity testing philosophy and a practical approach, as applied to pharmaceuticals, are described. Recently, there has been an effort to integrate the rodent (primarily rat) micronucleus assay with routine 2–4-week toxicokinetic studies. This approach has several advantages: 1) it utilizes the general principles of toxicology that govern the overall toxicity profile of a test substance; 2) factors such as the dose and/ or route of drug administration, drug metabolism, principles of toxicokinetics, and saturation of defense mechanisms are considered in evaluating genotoxicity; 3) it uses the concept of administering multiple tolerable doses aiding in achieving steady state plasma drug levels, which is more relevant for risk assessment compared to high acute doses; and 4) it helps minimize the amount of drug, number of animals used, and other resources. This integration approach can be extended to other toxicology studies and other relevant genotoxicity endpoints may be assessed. Based on the experience in our laboratory, integrating micronucleus assessment in routine toxicology testing is promising and should be utilized when practical. Environ. Mol. Mutagen. 32:115–120, 1998 © 1998 Wiley-Liss, Inc.     

Clinical, cytogenetic and molecular investigations in three patients with Wolf-Hirschhorn syndrome

Clinical, cytogenetic and molecular studies were performed in three patients with Wolf-Hirschhorn syndrome (WHS). In all cases the altered chromosome 4 appeared to be the result of a de novo deletion. Cytogenetic investigations located the breakpoint at 4p15.3 and 4p13. With cytogenetic methods it was not possible to decide whether these deletions were terminal or interstitial. DNA methods also failed to define a distal breakpoint within the 4p16.3 region which might have indicated an interstitial deletion. According to the literature, the paternal chromosome 4 is preferentially deleted in most patients with WHS. DNA analysis with polymorphic markers out of the 4p16.3 region revealed that in two of the cases reported here the deleted segment was of paternal and in one case of maternal origin.     

Molecular cytogenetic studies of Xq critical regions in premature ovarian failure patients

BACKGROUND: Premature ovarian failure (POF) is defined as amenorrhoea for more than 6 months, occurring before the age of 40, with an FSH serum level higher than 40 mIU/ml. Cytogenetically visible rearrangements of the X chromosome are associated with POF. Our hypothesis was that cryptic Xq chromosomal rearrangements could be an important etiological contributor of POF. METHODS: Ninety POF women were recruited and compared to 20 control women. Peripheral blood samples were collected and metaphase chromosomes were prepared using standard cytogenetic methods. To detect Xq chromosomal micro-rearrangements, fluorescence in situ hybridization (FISH) analysis was performed using a selection of 30 bacterial artificial chromosome (BAC) and P1 artificial chromosome clones, spanning Xq13-q27. We further localized the translocation breakpoints by FISH with additional BAC clones. RESULTS: Chromosomal abnormalities were identified in 8.8% of our 90 patients [one triple X, three large Xq deletions 46,X,del(X)(q22.3), 46,X,del(X)(q21.2) and 46,X,del(X)(q21.32), two balanced X;autosome translocations 46,X,t(X;1) (q21.1;q32) and 46,X,t(X;9)(q21.31;q21.2) and two Robertsonian translocations 45,XX,der(15;22)(q10;q10) and 45,XX,der(14;21)(q10;q10)]. The two Xq translocation breakpoints were among a cluster of repetitive elements without any known genes. FISH analysis did not reveal any Xq chromosomal micro-rearrangement. CONCLUSIONS: Karyotyping is definitely helpful in the evaluation of POF patients. No submicroscopic chromosomal rearrangements affecting Xq region were identified. Further analysis using DNA microarrays should help delineate Xq regions involved in POF.     

Cytogenetic studies in couples experiencing repeated pregnancy losses

A computerized database generated from the literature on cytogeneticstudies in couples experiencing repeated pregnancy losses hasbeen set up at the University of Quebec at Chicoutimi. At thepresent time, it contains data on 22 199 couples (44 398 individuals).The statistical analyses showed a relationship between the distributionof the chromosome abnormalities and the number of abortions.An uneven distribution of the chromosomal structural rearrangementsaccording to the sex of the carrier was found (P < 0.05).Overall, 4.7% of the couples ascertained for two or more spontaneousabortions included one carrier. It also appeared that only translations(both reciprocal and Robertsonian) and inversions were associatedwith a higher risk of pregnancy wastage. Therefore, geneticcounselling should be offered to these couples and investigationsperformed on their extended families.     

A cytogenetic survey of 14,835 consecutive liveborns

The results of chromosome studies on cultured umbilical cord blood lymphocytes from a consecutive series of 14,835 liveborn infants (7,608 males and 7,227 females) are described. Ninety-three infants (6.27 per 1,000) had a major chromosome abnormality. Of these, thirty-one infants (2.09 per 1,000) had sex chromosome abnormalities. Seven male infants had a 47,XXY karyotype, five had a 47,XYY karyotype, and three were mosaics. One male had a ring Y chromosome in all cells examined. A pericentric inversion of the Y chromosome was found in one case. Seven female infants had a 47,XXX karyotype, one had a 45,X karyotype and six were mosaics. Sixty-two infants (4.18 per 1,000) had autosomal abnormalities. There were twenty-one infants with trisomy 21 including one mosaic, six infants with trisomy 18, and two infants with trisomy 13 of a Robertsonian translocation type. Three infants had an unbalanced derivative chromosome resulting from a parental reciprocal translocation. Two infants with a partial monosomy of chromosome 13 were detected. There were four infants carrying an additional small marker chromosome. Twenty-four infants (1.62 per 1,000) had a balanced structural rearrangement of the autosomes; eleven with a Robertsonian translocation, eleven with a reciprocal translocation, and two with a pericentric inversion. The incidence of each type of major chromosome abnormality in this study was quite similar to that obtained from previous newborn surveys.     

Results of cytogenetic analysis in men with severe subfertility prior to intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) is increasingly [>ecomingthe treatment of choice for severe male subfertil-ity. Cytogeneticevaluation of men with andrological subfer-tility reveals anincreased incidence of chromosomal abnormalities when comparedwith the normal population. We performed chromosomal analysison the male partners of 32 couples referred for andrologicalsubfertility. In two of these men, constitutional chromosomaltranslocations were diagnosed prior to ICSI [(45, XY, t(21,-22)(pll; qll) and 46, XY, t(22; Y)(pll;ql2)]. Since ICSI bypassesmany potential barriers of fertilization, successful pregnancycan t>e achieved despite the presence of severely impairedspermatozoa in a population at high risk for chromosomal aberrations.It is well known that the presence of a chromosomal aberrationplays a significant role in partial >r complete spermatogenicarrest ICSI does not seem to increase the risk of fetal chromosomalabnormalities when a spermatozoon from a chromosomally normalmale is used. To exclude a higher risk for spontaneous abortionand fetal chromosomal abnormalities, we advocate cytogeneticscreening of males with severe male subfertility who opt forICSI.     

The minipig in toxicology

The use of pigs (Sus scrofa) in biomedical research is well established in particular in surgical and physiological research. For years both pigs and minipigs have been used in pharmacology and toxicology to answer specific questions when the more conventional species have been found unsuitable. The development of minipigs has resulted in strains of more manageable size than the domestic pig. Because of their well-accepted physiological and other similarities to humans, minipigs are becoming increasingly attractive toxicological and pharmacological models. There are several strains of minipigs (Göttingen, Yucatan, Sinclair, Hanford and other). This presentation is based on experience primarily with the Göttingen minipigs. In toxicology in Europe minipigs have become very popular for pharmaceutical studies in place of dogs and primates. Minipigs have been shown to be sensitive to a wide variety of drugs and chemicals. It has become obvious that minipigs can be used for all routes of administration, and in many cases are preferable to dogs or primates for metabolic or pharmacological reasons. There are advantages over the traditional non-rodent species in relation to specific responses to particular drug classes. Their use in general toxicology testing employing the continuous intravenous infusion, dermal or inhalation route has been described in detail in the literature. Background data on toxicological endpoints (ophthalmology, clinical pathology, ECG, organ weight, histopathology and reproduction parameters) have been well-established allowing studies to be interpreted. In the context of this conference, histopathology and toxicopathology data of spontaneous or drug-induced origin are available in the scientific literature. Now there is good supply of high-quality minipigs of known disease status. There are advantages over the traditional non-rodent species in relation to the ethical difficulties of use of animals in biomedical research. Consequently, there are scientific, economic and sociological reasons that make minipigs good toxicological and pharmacological models. The principal disadvantage is that toxicity testing in minipigs may require more test compound than the traditional species.     

恶性髓系血液病-7/7q-异常的分子细胞遗传学分析

目的 分析染色体-7／7q-在骨髓增生异常综合征(myelodysplastic syndrome，MDS)和急性髓细胞白血病(acute myeloblastic leukemia，AMI，)中的发生频率；探讨荧光原位杂交技术(fluorescence in situ hybridization，FISH)在检测和鉴定-7／7q-异常中的价值。方法 回顾性分析所有接受细胞遗传学分析(conventional cytogenetic analysis，CCA)的MDS／AML患者的核型特征，其中70份进行FISH分析。应用双色荧光直接标记的7号着丝粒探针(CEP7，光谱绿)和7q31基因序列探针(D7S486，光谱桔红)，15份正常样本作为对照。结果 -7／7q-在AML和MDS中出现频率分别为4．5l％(31／687例)和5．7l％(28／490例)，分别占异常核型病例的5．68％和l0．29％。7q-常见的缺失区域为7q21—22(10例)和7q31—35(10例)。FISH证实伴有克隆性-7／7q-异常，但在随机性-7／7q-异常或正常核型中未检出-7／7q-异常。在核型分析出现7q-异常的病例中，FISH检出7／11例可同时伴有-7克隆的出现，而且7q-异常的细胞数显著高于-7异常细胞数(42．5％vs8．4％，P=0．025)。1例核型为del(7)(q22)患者FISH证实为染色体易位；1例7q 患者FISH显示dup(7q)；1例复杂异常核型，FISH确定其累及7q。结论 FISH是鉴定或确定7q结构异常的强有力工具，能精确地评价-7／7q-。7q-异常通常与-7异常在同一个样本中共存，且7q-细胞数显著增高，推测-7克隆衍生于7q-的丢失。     

Prenatal cytogenetic diagnosis: First 1,000 successful cases

From February 1969 to August 1976, we studied 1,048 amniotic fluids. Of these, 958 (91.4%) were primarily for prenatal cytogenetic diagnosis. Cytogenetic studies were attempted in 1,021 cases; the diagnosis was successful in 1,000 of these. The failure rate of obtaining a diagnosis from the amniotic fluid cell culture of the first amniocentesis was 5% (50 cases); 29 cases had a repeat tap and successful diagnosis was achieved in all. In 21 cases, a repeat tap was refused. Thus, the overall failure rate of obtaining a final cytogenetic diagnosis was 2.06% (21/1,021). There were 32 fetal losses after amniocentesis including 16 spontaneous second trimester abortions, 7 fetal deaths in utero and 9 stillbirths. In two additional cases, fetal death had occurred before amniocentesis. This number of fetal losses does not exceed the number that would be expected in the same maternal age group without amniocentesis. In our series, the frequencies of trisomy in maternal age groups 40 years and over, 37–39 years, 35–36 years, and under 35 years were 4.5, 3.14, 0 and 0% respectively. These frequencies are comparable to those reported from other prospective prenatal studies and higher than those of retrospective live born studies. Various problems and pitfalls in prenatal cytogenetic diagnosis are discussed.     

The toxicology and immunology of detergent enzymes

Detergent enzymes have a very good safety profile, with almost no capacity to generate adverse acute or chronic responses in humans. The exceptions are the limited ability of some proteases to produce irritating effects at high concentrations, and the intrinsic potential of these bacterial and fungal proteins to act as respiratory sensitizers, demonstrated in humans during the early phase of the industrial use of enzymes during the 1960s and 1970s. How enzymes generate these responses are beginning to become a little clearer, with a developing appreciation of the cell surface mechanism(s) by which the enzymatic activity promotes the T-helper (T_H)-2 cell responses, leading to the generation of IgE. It is a reasonable assumption that the majority of enzyme proteins possess this intrinsic hazard. However, toxicological methods for characterizing further the respiratory sensitization hazard of individual enzymes remains a problematic area, with the consequence that the information feeding into risk assessment/management, although sufficient, is limited. Most of this information was in the past generated in animal models and in vitro immunoassays that assess immunological cross-reactivity. Ultimately, by understanding more fully the mechanisms which drive the IgE response to enzymes, it will be possible to develop better methods for hazard characterization and consequently for risk assessment and management.     

The toxicology properties of modified hydrothermal nanotitania extraction

IntroductionThe nanoparticle has become a part of world industry. This substance has been proven as potentially beneficial for its usage as a catalyst and semi-conductor due to its high surface area and the effects of the quantum size effect. It exhibits potential characteristics and would be applied in a wider scope of usage compared to bulk particles because the smaller the size of the particles, the more room for the extent of their usage. Nano titanium dioxide application as semi-conductors together with a catalyst is highly attributed to its high photochemical stability and ability to be produced at a low-cost. The consequence of this – exposure of nano titanium dioxide particles to humans – raises concerns regarding health and safety. Therefore, this research action works designed to offer a thorough analysis of toxicology impacts produced by our own synthesis modified hydrothermal in vitro experiments.Material and methodsOur nanotitania extraction with 0.05% silver was tested for its toxicity against L929 mouse cells. The cytotoxicity effect of nanotitania extract was evaluated by MTT assay. Cell viability (% CV) was calculated using a formula.ResultsThere are non-cytotoxicity activity of 0.05% nanotitania at concentrations 1.5, 3.1, 6.3, 12.5, and 25 mg/ml on L929 cell lines except at concentration 50 and 100 mg/ml. The result was related to the optical density reading.ConclusionsThere is no cytotoxic effect of nanotitania extraction with 0.05% silver in the growth inhibition test with L929 mouse with the exception of the 100 mg/ml extract.     

Non-culture based assays for the detection of fungal pathogens

Traditional, culture based methods for the diagnosis of fungal infections are still considered as gold standard, but they are time consuming and low sensitive. Therefore, in order to overcome the limitations, many researchers have focused on the development of new immunological and molecular based rapid assays that could enable early diagnosis of infection and accurate identification of fungal pathogens causing superficial and invasive infection. In this brief review, we highlighted the advantages and disadvantages of conventional diagnostic methods and possibility of non-culture based assays in diagnosis of superficial fungal infections and presented the overview on currently available immunochromatographic assays as well as availability of biomarkers detection by immunodiagnostic procedures in prompt and accurate diagnosis of invasive fungal infections. In addition, we presented diagnostic efficiency of currently available molecular panels and researches in this area.     

A clinical, cytogenetic and molecular study of ten probands with supernumerary inv dup (15) marker chromosomes

Ten probands with moderate to severe developmental delay were found to have a supernumerary inv dup (15) chromosome. These patients and their families were studied by both cytogenetic and molecular methods. Cytogenetic polymorphisms associated with the 15p short arm suggested a maternal derivation for the marker chromosome in all informative cases. One marker was directly maternally inherited. Molecular analysis employing Southern blotting and polymerase chain reaction (PCR) of microsatellite repeats demonstrated the presence of extra alleles in the 15q11q13 region. All ten of the probands demonstrated an extra band at one or more locus without recourse to densitometry. All of the inv dup (15) markers were comparable in size to a G group chromosome but there were differences in the positions of the breakpoints in 15q. There was an inconsistent relationship between marker size, gene dosage and severity of phenotype.     

Functional activities of synthetic anaphylatoxic peptides in widely used biological assays.

A comparison study was carried out between the modern ATP release assay (ARA) with guinea-pig platelets and the traditional guinea-pig ileum contraction assay (ICA). The biological activities of the anaphylatoxin C3a and synthetic C3a analogue peptides were determined in both assays. In dose-response curves with C3a, a human C3a peptide with the last 21 amino acids of the C terminus (C3a 56-77) and a peptide with 13 amino acids which was acylated N-terminal with the aromatic fluorenylmethoxycarbonyl group and an aminohexanoyl group (Fmoc-Ahx YRRGRAAALGLAR) were tested. The ARA turned out to be 100 times more sensitive than the ICA. In contrast to previous reports the 21 amino acid long C3a analogue peptide did not exhibit full C3a activity but only 7% (ARA) or 12% (ICA). The potentiation of biological activity in the ARA by coupling non-peptide acyl-residues N terminal to peptide C3a analogues could be confirmed with Fmoc-Ahx-YRRGRAAALGLAR in the ICA. In addition, the tri-peptide Fmoc-Ahx-LAR displayed C3a specific activity in the ICA demonstrated by desensitization experiments.     

Incidence and prognostic impact of cytogenetic aberrations in patients with systemic mastocytosis

The clinical behavior of systemic mastocytosis (SM) is strongly associated with activating mutations in KIT (D816V in >80% of cases), with the severity of the phenotype influenced by additional somatic mutations, for example, in SRSF2, ASXL1, or RUNX1. Complex molecular profiles are frequently associated with the presence of an associated hematologic neoplasm (AHN) and an unfavorable clinical outcome. However, little is known about the incidence and prognostic impact of cytogenetic aberrations. We analyzed cytogenetic and molecular characteristics of 109 patients (KIT D816V+, n = 102, 94%) with indolent (ISM, n = 26) and advanced SM (n = 83) with (n = 73, 88%) or without AHN. An aberrant karyotype was identified in SM‐AHN (16/73, 22%) patients only. In patients with an aberrant karyotype, additional somatic mutations were identified in 12/16 (75%) patients. Seven of 10 (70%) patients with a poor‐risk karyotype, for example, monosomy 7 or complex karyotype, and 1/6 (17%) patients with a good‐risk karyotype progressed to secondary acute myeloid leukemia (n = 7) or mast cell leukemia (n = 1) within a median of 40 months (range 2‐190, P = .04). In advanced SM, the median overall survival (OS) of poor‐risk karyotype patients was significantly shorter than in good‐risk/normal karyotype patients (4 vs 39 months; hazard ratio 11.7, 95% CI 5.0‐27.3; P < .0001). Additionally, the shortened OS in patients with poor‐risk karyotype was independent from the mutation status. In summary, a poor‐risk karyotype is an independent prognostic variable in advanced SM. Cytogenetic and molecular analyses should be routinely performed in all patients with advanced SM ± AHN because these investigations greatly support prognostication and treatment decisions.     

Endocrine toxicology: A Review of the application of endocrinology in experimental toxicology

In the past endocrine toxicology has not been a common subject in routine toxicity studies. However, since the endocrine system is an important integrating system of the body, controlling the major physiological functions, it is important to investigate the mechanism of action of exogenous compounds in endocrine target organs or hormonal target cells. The following procedure is suggested to detect effects on the endocrine system in routine toxicity experiments: (1) determination of the weight of endocrine organs and histology as screening parameters; (2) determination of circulating hormones in combination with morphological or immunocytochemical methods: (3) specific function tests and in vitro methods to determine dysfunction of specific endocrine organs or cells. That the use of such an approach has provided insight into the mechanism of action of chemical compounds will be demonstrated by results of endocrine toxicity studies with the antibiotic compound sulphadimidine, interfering with thyroid hormone synthesis as a secondary mechanism leading to thyroid tumour formation, the androgenic compound trenbolone acetate, used for growth promotion, for which the disturbance of the gonadal function formed the basis for the establishment of the no-observed-hormonal-effect level, the antibacterial compound furazolidone, suspected of having an oestrogenic activity which was hypothesized as the underlying mechanism for the observed mammary tumour formation, and the antimicrobial agent carbadox, used as feed additive for pigs, for which the interference with adrenal function, resulted in a severe disturbance of the water and salt balance in target animals. Originally presented at ECCP 93.