New methods for assessing male germ line mutations in humans and genetic risks in their offspring

Germ line mutations resulting from chemical or radiation exposure are a particular problem in toxicology as they affect not only the exposed generation but also an infinite number of generations thereafter. Established methods to show that these mutations occur in an F1 or subsequent population require the use of a large number of progeny for statistical significance. Consequently, many thousands of animals have been used in the past. Such a use is no longer considered desirable and is also very expensive. Several new molecular techniques (including analysis of tandem repeats and randomly amplified polymorphic DNA) now provide alternative methods of assessment, which also allow the quantification of individual mutations in individual sperm cells. These can also be applied to human offspring, making extrapolation obsolete. The downside of these methods is that they effectively determine the mutation rate in certain regions of DNA and the relevance of these to diseases, particularly cancer, is not always apparent. Therefore, it must be assumed that an increase in mutation rates in these selected regions correlates with altered phenotype. However, disease types linked to changes in tandem repeat length indicate that these may act as relevant markers for the development of phenotypes. Further research and evaluation are required to more closely link changes in DNA with altered phenotype and validate the use of tandem repeats and randomly amplified polymorphic DNA in transgenerational genotoxicity testing. This paper introduces and compares recently developed methods to assess mutations in sperm due to stem cell damage.     

The gastrocolic trunk of Henle in pancreatic surgery: an anatomo-clinical study

From 37 peroperative and cadaver anatomical investigations, it was concluded that, in contrast to the information in common texts on anatomy and surgery, a venous gastrocolic trunk was observed in only 46% of subjects; a true bipod gastrocolic trunk of Henle was a rare (8%) phenomenon. In this respect, a variate venous anatomy at the inferior border of the neck of the pancreas, as observed in this study, must be taken into account during pancreatic surgery and radiological procedures in the pancreas.     

A magnetic evaluation of peripheral nerve regeneration: I. The discrepancy between magnetic and histologic data from the proximal segment

Histologic techniques can quantify the number of axons in a nerve, but give no information about electrical conductibility. The number of functional myelinated neuronal units in a nerve can be quantified based on a magnetic recording technique. When studying reconstructed peripheral nerves a significant difference between the results found with these two techniques can be observed. A comparison was made between the long-term changes in the number of histologically and magnetoneurophysiologically measured neuronal units proximal to a nerve reconstruction. This study was performed on 6 New Zealand White rabbits, 20 weeks after the peroneal nerve had been reconstructed. The contralateral nerves were used as a control. Histologic examination demonstrates a statistically significant decrease of approximately 5% in the number of myelinated fibers. The magnetoneurophysiological results demonstrate a decrease which is estimated to be caused by the loss of approximately 50% of the functional myelinated neuronal units in the nerve. Therefore we conclude that of the initially available myelinated neuronal units, 5% degenerate completely, 45% are vital but lose their signal conducting capability, and the remaining 50% are vital and continue to conduct signals. Apparently, only this latter group of 50% of the initially available functional neuronal units appears to remain available for functional recovery. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:739–749, 1998.     

Effects of plant sterol and stanol ester consumption on lipid metabolism, antioxidant status and markers of oxidative stress, endothelial function and low-grade inflammation in patients on current statin treatment

OBJECTIVE: The present study was designed to examine for the first time, side-by-side, the effects of plant sterol and stanol consumption on lipid metabolism and markers of antioxidant status, oxidative stress, endothelial dysfunction and low-grade inflammation in subjects on stable statin-treatment. DESIGN: Double-blind, randomized, placebo-controlled, intervention trial. SETTING: University. SUBJECTS: Forty-five patients on current statin treatment were recruited via newspaper advertisements. Data of 41 patients were used in statistical analysis. INTERVENTION: Subjects consumed margarine with no added plant sterols or stanols for 4 weeks and were then divided into three groups of 15 subjects. For the next 16 weeks, one group continued with the control margarine and the other two groups with either a plant sterol- or stanol (2.5 g/day)-enriched margarine. Blood was sampled at the end of the run-in and intervention periods. RESULTS: Plant sterol and stanol consumption significantly (P=0.026) reduced low-density lipoprotein (LDL) cholesterol by 0.34 mmol/l (95% confidence interval (CI), -0.67 to -0.04 mmol/l). No effects were shown on enzymatic and non-enzymatic antioxidants and markers of oxidative modification of lipids and DNA. In addition, no effect was found on soluble adhesion molecules, C-reactive protein and monocyte chemotactic protein-1 concentrations. CONCLUSIONS: We conclude that 16 weeks of plant sterol or stanol consumption did not affect markers of antioxidant status, oxidative stress, endothelial dysfunction and low-grade inflammation in patients on stable statin treatment, despite a significant reduction of LDL cholesterol.     

A magnetic evaluation of peripheral nerve regeneration: II. The signal amplitude in the distal segment in relation to functional recovery

Motor and sensory function in a healthy nerve is strongly related to the number of neuronal units connecting to the distal target organs. In the regenerating nerve the amplitudes of magnetically recorded nerve compound action currents (NCACs) seem to relate to the number of functional neuronal units with larger diameters regenerating across the lesion. The goal of this experiment was to compare the signal amplitudes recorded from the distal segment of a reconstructed nerve to functional recovery. To this end, the peroneal nerves of 30 rabbits were unilaterally transected and reconstructed. After 6, 8, 12, 20, and 36 weeks of regeneration time the functional recovery was studied based on the toe-spread test, and the nerve regeneration based on the magnetically recorded NCACs. The results demonstrate that the signal amplitudes recorded magnetically from the reconstructed nerves increase in the first 12 weeks from 0% to 21% of the amplitudes recorded from the control nerves and from 21% to 25% in the following 23 weeks. The functional recovery increases from absent to good between the 8th and the 20th week after the reconstruction. A statistically significant relation was demonstrated between the signal amplitude and the functional recovery (P < 0.001). It is concluded that the magnetic recording technique can be used to evaluate the quality of a peripheral nerve reconstruction and seems to be able to predict, shortly after the reconstruction, the eventual functional recovery. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:750–755, 1998.     

Maternal intake of quercetin during gestation alters ex vivo benzo[a]pyrene metabolism and DNA adduct formation in adult offspring

Variation in xenobiotic metabolism cannot entirely be explained by genetic diversity in metabolic enzymes. We suggest that maternal diet during gestation can contribute to variation in metabolism by creating an in utero environment that shapes the offspring's defence against chemical carcinogens. Therefore, pregnant mice were supplemented with the natural aryl hydrocarbon receptor (AhR) agonist quercetin (1 mmol quercetin/kg feed) until delivery. Next, it was investigated whether the adult offspring at the age of 12 weeks had altered biotransformation of the environmental pollutant benzo[a]pyrene (B[a]P). In utero quercetin exposure resulted in significantly enhanced gene expression of Cyp1a1, Cyp1b1, Nqo1 and Ugt1a6 in liver of foetuses at Day 14.5 of gestation. Despite cessation of supplementation after delivery, altered gene expression persisted into adulthood, but in a tissue- and gender-dependent manner. Expression of Phase I enzymes (Cyp1a1 and Cyp1b1) was up-regulated in the liver of adult female mice in utero exposed to quercetin, whereas expression of Phase II enzymes (Gstp1, Nqo1 and Ugt1a6) was predominantly enhanced in the lung tissue of female mice. Epigenetic mechanisms may contribute to this adapted gene expression, as the repetitive elements (SINEB1) were hypomethylated in liver of female mice prenatally exposed to quercetin. Studies on ex vivo metabolism of B[a]P by lung and liver microsomes showed that the amount of B[a]P-9,10-dehydrodiol, B[a]P-7,8-dihydrodiol and 3-hydroxy-B[a]P did not change, but the amount of unmetabolised B[a]P was significantly lower after incubation with lung microsomes from offspring that received quercetin during gestation. Moreover, ex vivo B[a]P-induced DNA adduct formation was significantly lower for liver microsomes of offspring that were exposed to quercetin during gestation. These results suggest that prenatal diet leads to persistent alterations in Phase I and II enzymes of adult mice and may affect cancer risk.     

The drinking response of the chicken to peripheral and central administration of angiotensin 11

Intravenous injection of Ang II (val⁵ angiotensin II amide) elicited an immediate drinking response in the domestic fowl which lasted at least 20 minutes. The minimal dosage needed was 300 μg. Intracranial injection of 10 μg Ang II through cannulas implanted in the anterior diencephalon caused a significant increase in water intake. The minimal intracranial dosage of Ang II which evoked drinking was 2.5 μg. Intracranial injection of isotonic KCl inhibited the drinking response induced by intravenously injected Ang II when administered simultaneously. This suggests that drinking caused by both intravenous or intracranial injection of Ang II is activated through identical brain regions. The positive drinking response of the chicken to repeated consecutive intracranial injections of Ang II declined from the first injection through the following ones.     

The involvement of monkey premotor cortex neurones in preparation of visually cued arm movements

Some neurones in macaque postarcuate premotor area modulate their firing frequency in relation to motor tasks which require visual information. We previously reported that a large proportion of these neurones modulate during execution of a detour reaching task in which the movement phase was separated in time from the phase in which the monkey received a visual cue for the movement required to retrieve a food reward. A large proportion of task-related neurones (75%) modulated during this 'visual' phase, in which no task-related movements were made. This modulation was related to the position of the food reward, which served as the visual cue. Most of these neurones were located in cortical area 6, close to the arcuate curvature and its spur, but also more caudally in area 4 and rostrally in area 8. In the present chronic recording experiments in monkeys, several variations of the original task were used in order to test whether the 'visual'-related neuronal modulation could be involved in preparation of the upcoming movement. This modulation is unlikely to be related to any eye or arm movements occurring during the visual phase or to changes in environmental illumination. Neither can it be related to the presence of the visual cue in a particular part of the visual field, since the pattern of neuronal modulation was similar when a cue with a fixed position was used. This modulation was, however, contingent upon the occurrence of food retrieval during the subsequent 'movement phase', since it was abolished or diminished during presentation of a 'food-reward' which the monkey did not retrieve. For several neurones, modulation pattern during the visual phase depended on whether the food reward was to be retrieved with a gross hand movement or with relatively independent finger movements. It is likely, therefore, that neurones in the postarcuate premotor cortex are involved in preparation for arm movements with the help of visual cues. The results are discussed in view of corticocortical pathways which might be involved in transmission of visual information from visual areas through parietal association areas and premotor cortex to the primary motor cortex.     

Potential role of cytochrome P450‐1B1 in the metabolic activation of 4‐aminobiphenyl in humans

Metabolites of the human carcinogen 4‐aminobiphenyl (4‐ABP) form hemoglobin (Hb) adducts, which represent a useful biomarker for exposure. However, not every individual responds to a similar degree to 4‐ABP exposure, and variations in 4‐ABP‐Hb adduct formation might be explained by genetic polymorphisms in genes coding for enzymes involved in 4‐ABP metabolism. 4‐ABP‐Hb adducts were measured in blood samples from 57 smoking and 10 non‐smoking volunteers. An association was found between cigarette smoking and 4‐ABP‐Hb adduct levels in smokers (R² = 0.5, P < 0.001). Subsequently, subjects were genotyped for 12 polymorphisms in seven genes involved in biotransformation reactions. From this selection of polymorphisms, a significant impact was found for the CYP1B1 Leu⁴³²Val polymorphism (P = 0.021), which has been reported to lead to a decrease in enzyme activity. Indeed higher levels of 4‐ABP‐Hb adducts were observed in homo‐ and heterozygous carriers of the CYP1B1 ⁴³²Leu as compared to the double CYP1B1 ⁴³²Val genotype. A significant interaction between these CYP1B1 genotypes and the level of exposure was found (P = 0.003). Noteworthy, a saturation effect was observed for 4‐ABP‐Hb adduct formation at high smoking doses limited to carriers of the CYP1B1 ⁴³²Leu allele. No effect of polymorphisms in other genes were found. This is the first study in humans suggesting a crucial role of the CYP1B1 enzyme in 4‐ABP metabolism, indicating a protective effect of the CYP1B1 Leu⁴³²Val polymorphism against the formation of 4‐ABP‐Hb adduct levels, depending on the smoking dose. © 2009 Wiley‐Liss, Inc.