Studies of the induction of dominant lethals and translocations in male mice after chronic exposure to microwave radiation

Male C3H mice were exposed to 100 W m-2 of 2.45 GHz continuous-wave microwave radiation for 6 h per day for a total of 120 h over an 8-week period. The exposure level was chosen so that the specific energy absorption rate (SAR) would be approximately equal to the level of 4 W kg-1 which is considered by a number of organizations to be a threshold for adverse biological effects. At the end of the treatment period the mice were mated with a different group of (C3H x 101) F1 hybrid females each week for the following 8 weeks. There was no significant reduction in pregnancy rate, preimplantation survival or postimplantation survival in the exposed group compared to sham-exposed controls. At the end of the mating period a cytogenetic analysis was carried out of meiotic chromosome preparations of testicular tissue, thus sampling cells that were stem cell spermatogonia during the treatment regime. The results showed no difference in the frequency of reciprocal translocations between the sham and treated groups, or in the frequency of cells with autosome or sex chromosome univalents. Low levels of fragments and exchanges were found in both groups. It is concluded that there is no evidence in this experiment to show that chronic exposure of male mice to 2.45 GHz microwave radiation induces a mutagenic response in male germ cells. This conclusion is in agreement with the observations of Berman et al. (1980), who reported a lack of male germ cell mutagenesis after repetitive or chronic exposure of rats to 2.45 GHz.     

Heritable translocation study in male mice with trimethyl phosphate

Dominant lethal and heritable translocation studies were performed in male mice receiving a single intraperitoneal injection of trimethyl phosphate (TMP). The germ cell stage investigated was the spermatid. Methyl methanesulfonate (MMS) was used as a positive control in the latter study. A dominant lethal assay gave marked dose-dependent increases in early fetal deaths. Heritable translocations were detected at 1000 or 1500 mg of TMP/kg in F1 male progeny when screening for semi-sterility and cytogenetically analyzing the meiotic or mitotic chromosomes. Translocation induction was higher at the higher TMP dose (14.3%) than at the lower dose (5.3%) and the yield from the higher dose was similar to that induced by 50 mg of MMS/kg (11.0%). Most of the translocation carriers were semi-sterile or sterile. The data confirm conclusions from other dominant lethal studies showing TMP to be capable of causing chromosomal damage in mouse spermatids and show that certain types of damage result in heritable translocations.     

Strategies for detecting heritable translocations in male mice by fertility testing

Data from a heritable translocation test were analysed to estimate the best rule for classification of F₁ males in normals or partially sterile translocation carriers according to litter size or numbers of live and dead implants per mating. Six rules were com[ared for classification with up to three litter sizes per F₁ male observed. The results indicate that a translocation rate of 2%, or at best of 1%, can be detected with reasonable cost.     

Strategies for detecting heritable translocations in male mice by fertility testing.

Data from a heritable translocation test were analysed to estimate the best rule for classification of F1 males in normals or partially sterile translocation carriers according to litter size or numbers of live and dead implants per mating. Six rules were compared for classification with up to three litter sizes per F1 male observed. The results indicate that a translocation rate of 2%, or at best of 1%, can be detected with reasonable cost.     

Synaptonemal complex analysis of X-7 translocations in male mice

The synaptonemal complexes of surface-spread spermatocytes of mice heterozygous for one of two reciprocal translations (R3 and R5) between the X and chromosome 7 have been examined by light and electron microscopy (EM). The break points of R3 were determined to be at 70% of chromosome 7, as measured from the centromere, and at 22% of the X. Translocation quadrivalents were formed almost exclusively. The break points of R5 were at 21% of chromosome 7 as measured from the centromere, and at 83% of the X. There was little indication that the break in the X interfered with sex-chromosome synapsis between the 7X and Y. Univalent Y's were not observed in R3, and only seldom observed (8–14%) in R5. However, in contrast to R3, R5 formed quadrivalents relatively rarely (20% in the EM study of 100 nuclei), and heteromorphic bivalents of 7X-Y and X7-7 quite frequently (72%). Possible causes of this high bivalent frequency are discussed. Light-microscope (LM) analysis alone was found to be inadequate for interpreting synaptic configurations (quadrivalents vs. bivalents) in R5. The LM analysis was further complicated by the occurrence of nonhomologous synapsis in the heteromorphic bivalents of R5, a phenomenon easily recognized and interpreted in the EM portion of the study.     

Calculations of dietary exposure to acrylamide

In this paper we calculated the usual and acute exposure to acrylamide (AA) in the Dutch population and young children (1–6 years). For this AA levels of different food groups were used as collected by the Institute for Reference Materials and Measurements (IRMM) of the European Commission's Directorate General Joint Research Centre (JRC) from April 2003 up to May 2004. This database contained about 3500 AA levels received from mainly Germany, The Netherlands, Ireland, Greece, Austria, UK and from food industry. Food consumption levels used were derived from the Dutch National Food Consumption Survey of 1997/1998 (n = 6250 of which 530 children aged 1–6 years). The exposure was estimated using the probabilistic approach. The results of the exposure calculations are discussed in relation to different methodological aspects of AA exposure calculations and possible uncertainties related to this. The items discussed include quality of the AA levels measured in food items, the allocation of AA levels to food categories, the quality of food consumption levels, and relevant exposure model in relation to reported toxicity of AA. Furthermore, we demonstrate that scenario studies and probabilistic modelling of exposure are potential useful tools to evaluate the effect of processing techniques to reduce AA levels in food on AA exposure. The scenarios studied reduced total AA exposure ranging from <1% up to 17%.     

Calculations of dietary exposure to acrylamide

In this paper we calculated the usual and acute exposure to acrylamide (AA) in the Dutch population and young children (1-6 years). For this AA levels of different food groups were used as collected by the Institute for Reference Materials and Measurements (IRMM) of the European Commission's Directorate General Joint Research Centre (JRC) from April 2003 up to May 2004. This database contained about 3500 AA levels received from mainly Germany, The Netherlands, Ireland, Greece, Austria, UK and from food industry. Food consumption levels used were derived from the Dutch National Food Consumption Survey of 1997/1998 (n=6250 of which 530 children aged 1-6 years). The exposure was estimated using the probabilistic approach. The results of the exposure calculations are discussed in relation to different methodological aspects of AA exposure calculations and possible uncertainties related to this. The items discussed include quality of the AA levels measured in food items, the allocation of AA levels to food categories, the quality of food consumption levels, and relevant exposure model in relation to reported toxicity of AA. Furthermore, we demonstrate that scenario studies and probabilistic modelling of exposure are potential useful tools to evaluate the effect of processing techniques to reduce AA levels in food on AA exposure. The scenarios studied reduced total AA exposure ranging from <1% up to 17%.     

Stress induced in heart and other tissues by rat dermal exposure to JP-8 fuel

Limited information is available regarding the development of systemic organ stress by dermal exposure to JP-8 fuel. In this study, the systemic stress potential of this fuel is evaluated in a rat model subjected to dermal applications of JP-8 for 7 days at 300 μl per day. Tissue histology indicated that JP-8 induces morphological alterations that suggest that tissue stress in the heart is more substantial than stress in the kidney and liver. Immunoblot analysis of tissues revealed increased levels of the inducible heat shock protein 70 (HSP70) in the heart, kidney, and liver after this dermal JP-8 exposure. This exposure also leads to increased levels of heme oxygenase-1 (HO-1/HSP3) in the liver. Additionally during this exposure, a negative regulator of inflammation, IκBα (inhibitor of NF-κB), was increased in the liver, slightly increased in the kidney, and not increased in the heart. Two regions of the rat brain were also examined and HSP70 and IκBα were increased in the cerebellum but not significantly increased in the cortex. This study indicates dermal JP-8 exposure causes systemic alterations that are associated with cytoprotective activities (e.g., in the liver) as well as potentially toxic mechanisms (heart and kidney).     

Cytogenetic analysis of thioTEPA-induced translocations in the spermatids of male mice

Spermatocytes of 36 reciprocal translocation heterozygotes at diakinesis-MI stage were studied cytogenetically. There were 38.8 % cells with rings and 32.8 % - with chains and univalents among 3666 cells. It is concluded that intensive mutational process producing 30 % of translocation heterozygotes is characterized by random breakage along the chromosomes.     

Reciprocal translocations in germ cells of male mice receiving external gamma-irradiation

Data reported in the literature up to 1985 on reciprocal translocation induction in male mouse germ cells by external gamma-ray doses ranging from 0.5 to 6.0 Gy delivered at fixed dose rates were analyzed. On the assumption of a non-threshold linear dose response, zero effect at zero dose, and a center of distribution lying on an approximately straight line, calculations were made of linear regression coefficients. These coefficients (b), as a function of the dose rate (P), were well fitted by two straight lines: b = (3.15 +/- 0.59 log P) X 10(-6) for dose rates from 0.01 to 0.1 mGy/min; and b = (7.52 +/- 3.86 log P) X 10(-6) for dose rates ranging from 0.06 to 1.2 X 10(3) mGy/min. The intersection point of these two lines determined the so-called threshold level of the dose rate, namely, 4.6 X 10(-2) mGy/min, at which the effectiveness of external gamma-irradiation is not expected to exceed 2.36 X 10(-6)/mGy. In addition, experiments were undertaken in which yields were recorded of reciprocal translocations in germ cells of male mice exposed to 0.9 Gy of gamma-radiation at dose rates ranging from 6.14 X 10(-3) to 6.14 X 10(2) mGy/min (6 levels); comparisons were made with data published up to 1985 from similar studies using other fixed doses. To do this, translocation yields were expressed as relative yields (F) and their relationship to the dose rate (P) for the individual fixed doses was represented by an equation of the type: F = alpha + beta log P. For most of the equations, the regression coefficients were in good agreement and a single relationship was obtained to represent them. From the analysis performed it follows that, within the 0.6-6.0 Gy dose range, the pattern of the F vs. P relationship is unaffected by the dose. This supports the initial assumption that for the dose range up to 6.0 Gy the dose response for the reciprocal translocation yield is a non-threshold straight-line relationship.     

Transmission of X-ray-induced reciprocal translocations in normal male mice and in male mice with a reduced sperm count due to translocation homozygosity

Normal (+/+) and translocation T(1; 11.13S)70H homozygous (T/T) male mice received 2 X 2.5 Gy X-rays with a 24-h interval. After 120 days, the frequency of late diplotene-metaphase I spermatocytes with translocation multivalents was 14.1% for +/+ and 13.7% for T/T males, respectively, in one group of animals of each type. The difference is not significant. A second group was allowed to sire progeny for 60 days with 2 normal females per week. Reciprocal translocations detectable at diakinesis/metaphase I were observed in 2.5% of the 395 male progeny from the irradiated +/+ fathers, and in 2.9% of the 489 male progeny from the irradiated T/T fathers. This leads to a pooled estimated transmission of 0.81 +/- 0.19. Translocations induced in the long 11.13 metacentric chromosome were not transmitted with a different frequency. The rate of heritable induced translocations in this study was 5.4 X 10(-5)/rad/gamete. On the basis of the data of Generoso et al. (1984) for the frequency of the heritable spontaneous translocations in male mice, it is concluded that, because of their low doubling dose (3.3-4.6 rad), the spontaneous translocations are probably of postmeiotic origin.     

Chloroplast translocations induced by light pulses

The effect of single blue-light pulses on chloroplast rearrangement was studied in the leaves of Tradescantia albiflora, Chlorophytum elatum, and Lemna trisulca. For measuring translocations in terrestrial plants the method of transmission changes was used; translocations in the water plant Lemna were studied by direct microscopic observation and counting. Strong light (30 W m^-2) applied in the form of short pulses, shorter than a lag period of translocations, induces some transient effects in the following dark period. With short pulses, transient rearrangements of chloroplasts to a weak-light position were found. With longer pulse duration, biphasic responses took place in Tradescantia and Lemna: The initial movement to a partial strong-light position was followed by a wave of translocation to a weak-light arrangement. In Chlorophytum this type of response appeared only in a narrow fluence range. The validity of the reciprocity law in relation to fluence rate and time of irradiation was confirmed for Tradescantia. The results may give us an insight into the kinetics of the primary effects of light in the translocation process.     

Heritable chromosome aberrations in mammals after exposure to chemicals

Summary The observation of dividing spermatocytes is routinely used to detect the induction of heritable chromosome aberrations such as reciprocal translocations in the treated animals or in their F1 offspring. 37 compounds have so far been tested for the induction of chromosome rearrangements in spermatogonia. Only 9 gave positive results. However, positive results were observed for all alkylating agents in the F1 test. From these observations it can be concluded that the spermatogonia which are the main germ cell type at risk represent a relatively safe germ cell stage.     

Chromosome aberrations induced by monomeric acrylamide in bone marrow and germ cells of mice

Acrylamide induces chromatid exchanges and breaks with considerable frequency in spermatogonia of mice with long-term administration (3 weeks), though not, remarkably, with short-term administration (1–2 weeks). At 12 and 24 h after single injections with 50, 100 and 150 mg/kg acrylamide, evaluation of the cytogenetic effect is difficult in the spermatogonia because of an extreme reduction of mitotic cells. Aneuploid and polyploid cells increase with ti,e after treatment in both marrow and spermatogonial cells, while the aberration frequency shows no increase in marrow after both oral-administration and injection. Evidently the spermatogonia are thus rather more sensitive to acrylamide than marrow cells. On the other hand, the SCE frequency is at the control level in treated subjects in marrow and spermatogonia. Acrylamide induces chain quadrivalents, ring quadrivalents, fragments and univalents which are particularly evident in primary spermatocytes in both oral administration and injection, though it is questionable whether these structural changes deal with spermatogonia, or otherwise with the S-phase primary spermatocytes. There is a possibility that the aberrant cells thus produced can develop into spermatozoa carrying a certain type of reciprocal translocation which leads to semi-sterile progeny. In relation to the above problem detailed investigations into this type of rearrangement in primary spermatocytes are needed.     

Activin A and equine chorionic gonadotropin recover reproductive dysfunction induced by neonatal exposure to an estrogenic endocrine disruptor in adult male mice

We aimed to elucidate the mechanism of action of estrogenic endocrine disruptors and the rescue of reproductive function, particularly the responsiveness of testes to eCG and/or activin A (ACT) after establishing reproductive disorders. Newborn male mice (n = 29) were randomly divided into an untreated group and three treatment groups that received diethylstilbestrol (DES; 100 mug per animal) subcutaneously on Postnatal Day 3 to establish reproductive disorders and daily treatment with PBS (controls: DES + PBS), eCG (eCG group: DES + eCG), or eCG + ACT (eCG + ACT group: DES + eCG + ACT) at 6-8 wk of age prior to mating. After treatment, the controls showed diminished Leydig cells in the testes and thin germ cell layers containing pyknotic germ cells and multinucleated cells. In the eCG and eCG + ACT groups, spermatids and Leydig cells increased markedly. The immunoexpression of androgen receptors in the eCG group and steroidogenic acute regulatory (STAR) protein in the eCG and eCG + ACT groups recovered to approximately the levels in the untreated group; plasma LH and testosterone levels also increased relative to those in the controls. In addition, the cell proliferation index, which is estimated from 5-bromo-2'-deoxyuridine immunoexpression in spermatogonia, increased significantly under eCG treatment, and even more with eCG + ACT. However, the numbers of germ and Leydig cells decreased at 12 wk of age. Thus, ACT and eCG help the testes to recover from the dysfunction induced by neonatal DES administration. Furthermore, the permanent male reproductive disorder induced by neonatal exposure to estrogenic agents may be more likely to result from dysfunction of the hypothalamic-pituitary axis than from dysfunction of the lower reproductive organs.