Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation.

Exposure of spermatozoa to reactive oxygen species (ROS) has been associated with cellular injury, that includes DNA damage and lipid peroxidation. In addition, sperm preparation techniques such as centrifugation, commonly used prior to in vitro fertilization and scientific studies, are associated with the generation of ROS and an increase in the level of DNA damage. The preservation, therefore, of sperm in vitro that might decrease the potential for oxidative DNA damage to arise and allow for an improvement in semen quality used for artificial insemination, is of importance. Seminal plasma is a rich source of antioxidants, which, potentially, safeguards sperm from oxidative attack during storage and once ejaculated. We have investigated the protection of human spermatozoa from ROS afforded by seminal plasma. Sperm were exposed to exogenous ROS by incubating the cells with hydrogen peroxide in the presence of ferrous sulfate and ADP. Aliquots of seminal plasma were added to the incubation mixture in differing amounts, and the generation of DNA strand breaks and thiobarbituric acid reactive species (TBARS), indicative of lipid peroxidation, determined. Incubation of sperm with exogenous ROS resulted in a significant generation of DNA strand breaks and lipid peroxidation compared to basal levels of damage (P<0.05). Addition of seminal plasma to the incubation media produced a significant decrease in DNA strand breaks and TBARS (P<0. 05), when the amount of plasma added exceeded 60% v/v. The results indicate that spermatozoal oxidative damage induced by exogenous ROS, specifically DNA damage and lipid peroxidation, is reduced by the presence of seminal plasma.

An unusual helical micro-organism found in the gut lumen of human subjects.

An earlier report described the discovery of a micro-organism in the form of a double helix in human small bowel biopsies. Mucosal biopsies of the stomach and small bowel obtained from patients with rheumatic diseases and dyspepsia by enteroscopy and gastroscopy were fixed for scanning electron microscopy to investigate the organism further. In 62% of biopsies, an organism in the form of a double helix with bifid ends, 5-30 microm long, was found lying free on the surface of the mucosa. The organism has been demonstrated in the stomach, duodenum and small bowel. Flagella were never seen to be associated with the organism. In spite of its helical form, the organism lacks many of the factors associated with spirochaete morphology. It is suggested that this, as yet unnamed organism, may be found throughout the length of the digestive tract. Its pathological significance is not known.

Antioxidant capacity of the epididymis.

The human epididymis provides an optimal environment for the storage and maturation of spermatozoa. However, the ability of the epididymis to protect spermatozoa from oxidative attack whilst stored at this site, through the local actions of antioxidants, has not thus far been well studied. This study assessed the contribution of the epididymis to seminal plasma antioxidant activity, by comparing the semen of normozoospermic and vasectomized men. Total seminal plasma antioxidant activity was measured, as were concentrations of urate, ascorbate and thiols, antioxidants that are abundant in human semen. Thiobarbituric acid reactive species (TBARS) were measured to indicate lipid peroxidation. Total antioxidant activity and thiol content were significantly lower (P < 0.05) in the plasma from vasectomized men compared with that of normozoospermic donors. Ascorbate and urate were found at similar concentrations in the plasma of both groups. The concentration of TBARS was significantly higher (P < 0.001) in the semen from vasectomized individuals compared with the normozoospermic group. The results indicate that the epididymis contributes to the antioxidant capacity of seminal plasma and possesses region-specific antioxidant activity, which may potentially protect spermatozoa from oxidative attack during storage at this site.

Extra-epididymal spermatozoa express nuclear abnormalities.

Extra-epididymal spermatozoa account for approximately a third of all spermatozoa found in the normal human ejaculate. Whilst remaining outside of the testes at core body temperature, the functional competence of spermatozoa, including cell motility and fertilizing capacity, diminishes. By examining spermatozoa found in the seminal fluid of recently vasectomized men, this study has investigated the nuclear changes that occur in spermatozoa whilst persisting in sites distal to the epididymis. Spectral recordings of spermatozoa stained with the nucleic acid dye, toluidine blue and the sperm chromatin structure assay (SCSA) were performed. Toluidine blue staining of human sperm DNA is an effective predictor of abnormal protamine disulphide crosslinking and chromatin condensation. Using flow cytometry, the SCSA determines the sensitivity of sperm DNA to acid-induced denaturation, providing a measure of chromatin and DNA damage. Abnormal protamine disulphide crosslinking and chromatin condensation was significantly higher in spermatozoa from patients after vasectomy when compared to normozoospermic controls (p < 0.01). Additionally, spermatozoa from vasectomized donors were significantly more sensitive to acid-induced denaturation than were normozoospermic donors (p < 0.05). The results indicate that spermatozoa surviving in extra-epididymal sites are more likely to possess DNA and chromatin abnormalities than those present in the testes and epididymis. These changes may partly explain the depletion of cell viability and fertilizing capacity of extra-epididymal spermatozoa which has been reported previously.

Sperm chromatin damage associated with male smoking.

Cigarette smoke is a rich source of mutagens and carcinogens; thus, we have investigated the effects of male smoking on the DNA of human sperm. This was performed using the sperm chromatin structure assay (SCSA), which measures the sensitivity of sperm DNA to acid induced denaturation, and the terminal deoxynucleotidyl transferase assay (TdTA), which measures DNA strand breaks by addition of the biotinylated nucleotide dUTP to 3'-OH ends of DNA, sites of DNA breakage, using the enzyme terminal deoxynucleotidyl transferase. Sperm from subjects who smoked were significantly more sensitive to acid induced denaturation than non-smokers (P<0.02) and possessed higher levels of DNA strand breaks (P<0.05). We hypothesise that smoking damages the chromatin structure and produces endogenous DNA strand breaks in human sperm. These changes may result in sperm DNA mutations, that predispose offspring to greater risk of malformations, cancer and genetic diseases.

Rapid and simple chromatographic method for the determination of diazepam and its major metabolites in human plasma and urine.

A simple, rapid, sensitive and selective HPLC method has been developed for the analysis of diazepam (DZP) and its major metabolites, N-desmethyldiazepam (DMDZP), temazepam (TZP) and oxazepam (OZP), in plasma and urine, using clonazepam (CZP) as the internal standard and chloroform as the extracting solvent, with a 10 ng/ml limit of quantitation for the four assayed drugs, and an average (+/-S.D.) recovery of 87.7+/-6.46%, 92.9+/-5.31%, 91.4+/-4.01% and 91.7+/-2.68% for DZP, DMDZP, TZP and OZP, respectively (from plasma), and 89.6+/-2.26%, 90+/-4.24%, 87.45+/-0.64% and 94.50+/-0.71% for DZP, DMDZP, TZP and OZP, respectively (from urine). The method has also proved to be selective and reproducible.

Melanocortin probes for the melanoma MC1 receptor: synthesis, receptor binding and biological activity.

The fate of alpha-melanocyte-stimulating hormone (alpha-MSH) subsequent to binding to the melanoma melanocortin MC1 receptor is of interest with regard to its potential use in targeting cytotoxic drugs or imaging to melanoma. Tools such as iodinated, photoaffinity-labelled, biotinylated and fluorescent melanocortins are required to study the fate of the ligand during its interaction with the receptor. In this study, a series of probes for the receptor based on the potent analogue, [Nle4,Dphe7]alpha-MSH, have been developed and tested for their potential usefulness. All probes contain the core melanocortin motif His-Phe-Arg-Trp. They bind the receptor readily and appear to have similar intrinsic efficacies to the endogenous peptide, so that biological activity is regulated by their receptor binding affinity. Functional photoaffinity-labelled, biotinylated and fluorescent probes are described. The biotinylated probe binds the receptor when coupled to streptavidin, although with reduced affinity.

Caffeine as a metabolic probe: NAT2 phenotyping.

1. Caffeine has been used to determine acetylator phenotype for some 15 years but the interpretation of metabolic ratios with this substance raises theoretical and methodological issues. 2. N-acetyltransferase type 2 (NAT2) status was assessed in 23 young healthy subjects using both caffeine overnight and spot urine samples, and sulphadimidine. 3. Frequency distribution analysis of the metabolic ratios of NAT2, indicated two distinct groups for sulphadimidine, and for caffeine spot but not overnight samples. Spearman's rank correlation values were low indicating differences between the data sets for sulphadimidine and spot caffeine samples. Correlation between the two urine collection periods for caffeine was poor. 4. The complex metabolism of caffeine may compromise its value as a probe for determining acetylator phenotype until the effects of important variables are better understood.

Compulsory random drug testing of prisoners in England and Wales: design flaws in the system.

The Home Office has recently introduced compulsory testing of prisoners in England and Wales for drug abuse. From 1996 all prisons in the UK will be involved. Urine samples from approximately 10% of the prison population will be collected each month. The method of drug analysis selected by the Home Office is fast and economical but readily prone to interference from common substances giving false results. An elaborate procedure has therefore been evolved including a rigorous personal search of the prisoner to prevent sample adulteration. The definitive test gas chromotography - mass spectrometry (GC-MS), is more expensive but is resistant to sample adulteration, and currently all positive samples are confirmed by this method. In view of the proportion of samples that have tested positive, the extent of the unknown number of false negatives, and the possible rejection of the collection protocol by prisoners, savings could be made if the method of analysis employed in the first instance was GC-MS. This paper illustrates the inaccuracies produced in an assay technique similar to that used by the Home Office when urine is contaminated by simple, commonly available substances.

An in vitro technique for the rapid determination of drug entry into breast milk.

1. A simple in vitro technique that predicts drug transfer into breast milk is described. 2. Drugs of differing physical and chemical characteristics were tested. 3. The technique provides an experimental system for studying plasma to milk transfer with changing milk composition. 4. A mechanism proposing a role of milk proteins in controlling drug entry into milk is discussed.

Characteristics of ulcers of the small bowel induced by non-steroidal anti-inflammatory drugs in the rat: implications for clinical practice.

Small bowel ulcers were created in the rat after the oral administration of non-steroidal anti-inflammatory drugs (NSAIDs). Of the six NSAIDs tested, indomethacin and diclofenac alone were associated with such damage which did not occur in a simple dose-related fashion. Bacteria were observed by electron microscopy in an active state of division in the base of the ulcers. When grown aerobically these were shown to be strains of Escherichia coli and Proteus mirabilis. Anatomically, NSAID-induced ulcers were found throughout the length of the bowel although more abundant in the proximal half. In vivo and in vitro sensitivity to antibiotics suggested that in addition to the bacteria identified, anaerobic beta-lactamase-producing organisms also have an important role in ulcer production in this model. This rat model of NSAID-induced gut toxicity is discussed in relation to the human situation, particularly for patients who take NSAIDs and who have an iron-deficiency anaemia and blood in their faeces, but no lesions in either the upper or lower bowel.

Identification of a melanocortin receptor expressed by murine brain microvascular endothelial cells in culture.

Microvascular endothelial cells were isolated from the brains of C57 mice and cultured in selective growth media. The isolation and culture techniques employed in this study minimised the contamination by nonendothelial cells such as astrocytes, pericytes, and smooth muscle cells. Microvascular endothelial cells examined using phase contrast light microscopy grew as small colonies of spindle-shaped cells which merged together to form typical contact-inhibited monolayers. The endothelial origin of these cells was determined using several established characterisation techniques. Preliminary receptor binding studies at 4 degrees using [125I-Tyr2, Nle4, D-Phe7]alpha-melanocyte-stimulating hormone ([125I-Tyr2, Nle4, D-Phe7]alpha-MSH) suggested the possibility that melanocortin receptors were present on the surface of brain microvascular endothelial cells. Subsequent binding isotherms confirmed that a small population of high-affinity melanocortin receptors was expressed. The existence of a specific binding site for alpha-MSH was confirmed by photoaffinity labeling with the 4-(1-azi-2,2,2,-trifluoroethyl)benzoic acid (ATB) derivative, [125I-Tyr2, Nle4, D-Phe7, (ATB)-Lys11] alpha-MSH. SDS-PAGE analysis identified the presence of a specific band with a molecular mass of approximately 45 kDa, which was consistent with previous data on melanoma melanocortin receptors, and represented a ligand-receptor complex. This study suggests that a receptor for alpha-MSH is expressed on the extracellular surface of murine brain microvascular endothelial cells; however, the physiological role of this receptor is as yet unknown.

Caffeine phenotyping of cytochrome P4501A2, N-acetyltransferase, and xanthine oxidase in patients with familial adenomatous polyposis.

Patients with familial adenomatous polyposis (FAP) and age and sex matched controls were tested for cytochrome P4501A2 (CYP1A2), N-acetyltransferase, and xanthine oxidase activities using caffeine urinary metabolites as a discriminator. FAP patients showed significant underactivity of N-acetyltransferase (which inactivates some carcinogens) and significant overactivity of CYP1A2 (which activates some carcinogens). Xanthine oxidase activity, which can generate free radicals and cause cellular damage, was significantly increased in the FAP patients. All but one of the FAP patients had undergone colectomy. A separate group of six patients was therefore assessed before and at an average time of eight weeks after colectomy. No effect on enzyme activity was seen. The differences in enzyme activities detected in this study could produce an excess of active carcinogenic metabolites in the bile of FAP patients and contribute to the high risk for intestinal cancer in FAP.

Caffeine as a metabolic probe: a comparison of the metabolic ratios used to assess CYP1A2 activity.

1. Caffeine is widely used as an in vivo probe for CYP1A2; the distribution/activity of this enzyme is reported to be reflected by metabolic ratios. 2. Several metabolic ratios using different combinations of urinary metabolites have been used to measure CYP1A2, with varying conclusions on its distribution. 3. A mathematical comparison of five metabolic ratios claiming to reflect CYP1A2 activity was made using data from 237 healthy volunteers. 4. All five metabolic ratios were symmetrically distributed. The five ratios however, measured at least three different parameters, with no one ratio correlating exactly with any other. 5. Data in the literature claiming to measure CYP1A2 using caffeine may reflect other parameters. 6. The complex metabolism of caffeine together with different parameters controlling the renal clearance of each metabolite, makes the use of urinary metabolic ratios an inaccurate probe for assessing the distribution of CYP1A2 activity in populations.

Rapid method for the routine determination of caffeine and its metabolites by high-performance liquid chromatography.

Caffeine is a popular compound for phenotyping individuals for CYP4501A2, xanthine oxidase (XO) and N-acetyltransferase (NAT) utilising urinary metabolites. The analysis is complex since at least thirteen metabolites are excreted by man. Past methods have been less than satisfactory in that either not all the metabolites have been resolved and/or extractions selective for particular groups of metabolites are required prior to chromatography. We report a method for the rapid analysis of caffeine and metabolites in urine that negates the requirement for an extraction step, and also a method for plasma analysis.