The dose-response relationship at very low doses of acrylamide is linear in the flow cytometer-based mouse micronucleus assay

Acrylamide (AA) is genotoxic and has been classified as a probable human carcinogen. Human exposure to AA may be high by the consumption of starch-based food that has been treated at high temperature, e.g. potato chips and crisps. For risk assessment, extrapolation to the expected low doses to humans will be more reliable when data from low experimental doses can be used. We have registered the effects of a series of low doses in the sensitive flow cytometer-based micronucleus assay in mice, paying special attention to deviations from the expected linear dose-response function. Two experiments were performed with CBA mice, injected i.p. with different doses of AA. In one experiment the effects of 22 doses (two mice per dose) ranging from 0 to 100 mg/kg b.w. were studied. In the second experiment seven doses (five mice per dose) ranging from 0 to 30 mg/kg b.w. were used. In both experiments, a clear increase of the frequency of micronucleated erythrocytes was seen, already at the lowest doses used. The dose-response function was found to be linear with a tendency to have a steeper rise at the lowest doses. The low DNA content of the micronuclei indicated an absence of whole chromosomes, i.e. no aneugenic effect of AA.     

Using network dynamic fMRI for detection of epileptogenic foci

Background

Epilepsy is one of the most prevalent neurological disorders. It remains medically intractable for about one-third of patients with focal epilepsy, for whom precise localization of the epileptogenic zone responsible for seizure initiation may be critical for successful surgery. Existing fMRI literature points to widespread network disturbances in functional connectivity. Per previous scalp and intracranial EEG studies and consistent with excessive local synchronization during interictal discharges, we hypothesized that, relative to same regions in healthy controls, epileptogenic foci would exhibit less chaotic dynamics, identifiable via entropic analyses of resting state fMRI time series.

Methods

In order to first validate this hypothesis on a cohort of patients with known ground truth, here we test individuals with well-defined epileptogenic foci (left mesial temporal lobe epilepsy). We analyzed voxel-wise resting-state fMRI time-series using the autocorrelation function (ACF), an entropic measure of regulation and feedback, and performed follow-up seed-to-voxel functional connectivity analysis. Disruptions in connectivity of the region exhibiting abnormal dynamics were examined in relation to duration of epilepsy and patients’ cognitive performance using a delayed verbal memory recall task.

Results

ACF analysis revealed constrained (less chaotic) functional dynamics in left temporal lobe epilepsy patients, primarily localized to ipsilateral temporal pole, proximal to presumed focal points. Autocorrelation decay rates differentiated, with 100 % accuracy, between patients and healthy controls on a subject-by-subject basis within a leave-one-subject out classification framework. Regions identified via ACF analysis formed a less efficient network in patients, as compared to controls. Constrained dynamics were linked with locally increased and long-range decreased connectivity that, in turn, correlated significantly with impaired memory (local left temporal connectivity) and epilepsy duration (left temporal – posterior cingulate cortex connectivity).

Conclusions

Our current results suggest that data driven functional MRI methods that target network dynamics hold promise in providing clinically valuable tools for identification of epileptic regions.

     

The impact of folate status and folic acid supplementation on the micronucleus frequency in human erythrocytes

Folic acid has a well-documented stabilising effect on chromosomes. A correlation between folate status and chromosome stability in humans has been reported in studies that were restricted to certain subpopulations, e.g., folate-deficient persons. The goal of the present investigation was to clarify if there also is a correlation between folate status and chromosome stability among individuals without any folate deficiency. The method used here is the recently developed flow cytometry-based micronucleus assay in human transferrin-positive reticulocytes (MN-Trf-Ret). In a blood sample, separation of the very young reticulocytes from the mature erythrocytes makes this micronucleus assay possible. This investigation comprises three studies (cross-sectional, giving baseline data), two of which are connected to an intervention study. In the three cross-sectional studies (total number of subjects, 99) the frequency of MN-Trf-Ret (fMN-Trf-Ret) was measured and compared with the serum folate status. In two of the studies also serum homocysteine and Vitamin B12 were measured and compared with the baseline fMN-Trf-Ret. Combining the results from the three cross-sectional studies, a negative correlation between folate status and fMN-Trf-Ret was obtained (p<0.05). The goal of the intervention studies was to clarify if different nutritional supplementations had any effect on the fMN-Trf-Ret and the cell proliferation (percentage polychromatic erythrocytes, PCE). Each of the two studies involved two groups, one placebo and one supplemented group. In one of the studies the supplementation was folic acid, 1000 microg/day during 1 week (n=30, both sexes); in the other intervention study, folic acid (800 microg/day), B12 (20 microg/day) and B6 (4 mg/day) were taken during 1 week (n=29, both sexes). No significant difference in %PCE or fMN-Trf-Ret between the two groups was found in either of the two intervention studies.     

Differences in the frequency of micronucleated erythrocytes in humans in relation to consumption of fried carbohydrate-rich food

The aim of this study was to investigate if consumption of ordinary carbohydrate-rich food prepared in different ways has an impact on chromosome stability, i.e., on the formation of micronucleated young erythrocytes in humans. Twenty-four persons, divided into two groups, participated during 4 days in a semi-controlled food-consumption study. One group (low-heated-food-group, LowHF-group) consumed only food boiled in water (max 100 degrees C) and the other group (high-heated-food-group, HighHF-group) consumed preferentially strongly heated (fried) food. From each of the subjects, blood samples were drawn, before and after 4 days. The frequency (f) of micronucleated (MN) very young erythrocytes (transferrin-positive reticulocytes, Trf-Ret), fMNTrf-Ret, was determined, and the difference in the frequency, before and after the eating period, was calculated. The obtained mean differences for the two groups were compared. As an indicator of highly heated food the acrylamide (AA) content in part of the consumed foodstuffs was analysed by use of LC/MS-MS and the AA intake estimated. In the blood samples the hemoglobin-adduct levels from AA were analysed as a measure of the internal AA dose. The differences between the mean fMNTrf-Ret, before and after the eating period, were -0.15 per thousand for the LowHF-group and +0.17 per thousand for the HighHF-group, p<0.005 (t-test, one-tailed). The mean total AA intake in the HighHF-group during 4 days was estimated to about 3000+/-450microg per person. For the LowHF-group, the mean AA intake was low, 20+/-10microg per person. The lowest dose of AA that caused a significant increase of micronucleated erythrocytes in mice is more than a hundred times higher than the AA level in this study. Thus, it is unlikely that the exposure to AA is the major cause behind the observed difference. The answer is probably to be found in other compounds produced at the same time during heating of the food.     

Adenomatous polyposis coli influences micronuclei induction by PhIP and acrylamide in mouse erythrocytes

Micronucleus (MN) induction in erythrocytes of multiple intestinal neoplasia (Min) mice with heterozygous Apc mutation was measured after s.c. injections of acrylamide, glycidamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and colchicine, and compared with wild-type (wt) mice. Since Apc influences microtubule dynamics, we wanted to test whether Min-mice were more sensitive to the production of MN than wild-type mice. We also examined the effect of pre-treatment with cytosine β-D-arabinofuranoside (Ara C) and hydroxyurea, which inhibit ligation of DNA strand breaks in the repair of DNA adducts. All compounds induced a significant increase in MN in both strains of mice with the following potencies: acrylamide < glycidamide < PhIP. No difference in the induction of MN was seen between Min-mice and wt-mice exposed to acrylamide, glycidamide or colchicine without pre-treatment. However, in Min-mice, PhIP treatment induced much less MN than in wt-mice, with about four- and six-fold increase in MN in Min-mice and wt-mice, respectively. A reduced ability to repair PhIP adducts may be the reason for the lower induction of MN in Min-mice. Treatment with Ara C and hydroxyurea, to increase sensitivity, gave more than a four-fold increase in MN, but strongly reduced proliferation. Pre-treatment with Ara C and hydroxyurea made the Min-mice slightly more sensitive to MN induction by glycidamide compared to wt-mice. We conclude that Min-mice are less sensitive than wt-mice to MN induction by PhIP that forms bulky DNA adducts, while Min-mice and wt-mice are equally sensitive to MN induction by acrylamide and glycidamide that form DNA base adducts.     

Low dose effects of chemicals as assessed by the flow cytometric in vivo micronucleus assay

Using flow cytometric automation of the mouse in vivo, micronucleus assay increases the sensitivity of the test. This is achieved through a very large increase in the number of cells scored, by a factor of 100×, which in turn greatly reduces the sampling error. With this method, dose–response relationships of in vivo micronucleus induction for four model agents mitomycin C (MMC), diepoxybutane (DEB), cyclophosphamide (CPA), and colchicine (COL) were studied at low dose levels. For the three clastogens MMC, DEB and CPA, linear dose–response relationships were found over the dose ranges studied, even in the very low dose region (defined as the dose region where the frequency of micronucleated erythrocytes is less than twice the baseline frequency). This is consistent with the view that no threshold should exist for genotoxic agents which target DNA. For COL a dose range was found, in which the frequency of micronucleated erythrocytes did not increase with dose, possibly indicating an in vivo threshold. The flow cytometric in vivo micronucleus assay represents one possibility for in vivo low dose–response studies.     

Human Gender Differences in the Perception of Conspecific Alarm Chemosensory Cues

It has previously been established that, in threatening situations, animals use alarm pheromones to communicate danger. There is emerging evidence of analogous chemosensory “stress” cues in humans. For this study, we collected alarm and exercise sweat from “donors,” extracted it, pooled it and presented it to 16 unrelated “detector” subjects undergoing fMRI. The fMRI protocol consisted of four stimulus runs, with each combination of stimulus condition and donor gender represented four times. Because olfactory stimuli do not follow the canonical hemodynamic response, we used a model-free approach. We performed minimal preprocessing and worked directly with block-average time series and step-function estimates. We found that, while male stress sweat produced a comparably strong emotional response in both detector genders, female stress sweat produced a markedly stronger arousal in female than in male detectors. Our statistical tests pinpointed this gender-specificity to the right amygdala (strongest in the superficial nuclei). When comparing the olfactory bulb responses to the corresponding stimuli, we found no significant differences between male and female detectors. These imaging results complement existing behavioral evidence, by identifying whether gender differences in response to alarm chemosignals are initiated at the perceptual versus emotional level. Since we found no significant differences in the olfactory bulb (primary processing site for chemosensory signals in mammals), we infer that the specificity in responding to female fear is likely based on processing meaning, rather than strength, of chemosensory cues from each gender.     

The time-course of micronucleated polychromatic erythrocytes in mouse bone marrow and peripheral blood

The time-course of micronucleated polychromatic erythrocytes (MPCE) in mouse bone marrow and peripheral blood, induced by an acute 0.1 Gy dose of X-rays, was determined using flow cytometric analysis, which made frequent sampling possible and allowed use of a dose low enough not to affect erythroid cell proliferation. The frequency of MPCE (fMPCE) began to increase in the bone marrow at 10 h after irradiation and reached a maximum at 28 h after irradiation. In the peripheral blood fMPCE began to increase at 20 h after irradiation and peaked at about 40 h after irradiation. The time-course found is discussed on the basis of data on the differentiation of erythroid cells. The results indicate that the micronuclei registered in polychromatic erythrocytes may originate from lesions induced not only during the last cell cycle but also during earlier ones. After an acute dose of 1.0 Gy of X-rays the maximum fMPCE was delayed both in bone marrow and peripheral blood reflecting an effect on the cell cycle progression of erythroblasts.     

Extended exposure of adult and fetal mice to 50 Hz magnetic field does not increase the incidence of micronuclei in erythrocytes.

The flow cytometer-based micronucleus assay was used to study the effects on chromosomes in erythroid cells of CBA/Ca mice after extended exposure to 50 Hz magnetic field (MF), 14 microT, peak-to-peak (p-p). The study included two different experiments: (a) mice exposed in utero during 18 days of their prenatal stage, and (b) adult mice exposed for 18 days. In experiment (a) 35 days after exposure was terminated, peripheral blood was drawn from the mice exposed in utero to determine whether the exposure had a genotoxic effect on the pluripotent erythroid stem cells. About 200000 polychromatic erythrocytes (PCE) and 200000 normochromatic erythrocytes (NCE) were analysed from each of 20 exposed mice. The EMF exposure did not significantly change the frequency of micronucleated PCE or NCE in comparison with 20 sham-irradiated mice. There was no difference in the proportion of PCE between exposed and unexposed animals. Similarly, in experiment (b) no differences were seen between EMF exposed and unexposed adult mice when samples of peripheral blood were taken at the end of exposure and analyzed for micronuclei in PCE and NCE. The proportion of PCE was the same in both groups. The results indicate that exposure to EMF does not induce direct or indirect effects on chromosomes in erythroid cells expressed as increased levels of micronucleated erythrocytes of mice. No indications of delayed genetic effects were found.     

HCV NS5B polymerase-bound conformation of a soluble sulfonamide inhibitor by 2D transferred NOESY

HCV NS5B RNA-dependent RNA polymerase (NS5B) is essential for viral replication and is therefore considered a target for antiviral drug development. From our ongoing screening effort in the search for new anti-HCV agents, a novel inhibitor 1 with low microM activity against the HCV NS5B polymerase was identified. SAR analysis indicated the optimal substitution pattern required for activity, for example, carboxylic acid group at 2-position of thiophene ring. We describe the steps taken to identify and solve the bioactive conformation of derivative 6 through the use of the transferred NOE method (trNOE).