Evidence of oxidative stress and associated DNA damage, increased proliferative drive, and altered gene expression in rat liver produced by the cholangiocarcinogenic agent furan.

Furan is a potent cholangiocarcinogen in rat by an as yet undefined mechanism. The risk to man remains unclear. Using a time-course stop study design, we have investigated the potential of furan to induce oxidative stress and DNA damage associated with inflammatory and regenerative responses in rat liver. Furan was administered via oral gavage (30 mg/kg b.w. 5 daily doses per week), and livers were analyzed at time points between eight hr and three months. A one-month recovery group previously treated for three months was also included. There was a marked association between CYP2E1 expression and DNA oxidation (8-oxo-dG) in areas of centrilobular hepatocyte necrosis seen after a single dose. After one-month recovery from three-month treatment, 8-oxo-dG was still observed in areas of furan-induced cholangiofibrosis. Furan-induced changes in the expression of various genes associated with oxidative stress, DNA damage, and cell cycle control were identified during treatment and recovery. We propose that furan-induced cholangiocarcinomas emerge from areas of cholangiofibrosis as a result of a combination of chronic, persistent indirect damage to DNA through oxygen radicals coupled with persistent proliferative signals, including loss of connexin 32, that act to convert this DNA damage to fixed mutations.

Induction and progression of cholangiofibrosis in rat liver injured by oral administration of furan.

Cholangiofibrosis is a structural anomaly that precedes the development of cholangiocarcinoma in some rodent models. In this article, the authors examine the contribution of the epithelial and mesenchymal cells in the pathogenesis of this complex lesion. Furan was administered to rats by gavage in corn oil at 30 mg/kg b.w. (five daily doses per week) and livers were sampled between eight hr to three months. Characteristically the administration of furan caused centrilobular injury, and restoration was accomplished by proliferation of hepatocytes. Some areas of the liver were, however, more severely affected, and here, injury extended into portal and capsular areas, which resulted in a rapid proliferation of ductular cells that extended into the parenchyma accompanied by a subtype of liver fibroblasts. These ductules either differentiated into hepatocytes, with loss of the associated fibroblasts, or progressed to form tortuous ductular structures that replaced much of the parenchyma, leading to cholangiofibrosis. Although it is unclear what determines the difference in the hepatic response, a loss of micro-environmental cues that instigate hepatocyte differentiation and termination of the hepatocyte stem cell repair response may be perturbed by continual furan administration that results in an irreversible expansile lesion that may mimic the features of cholangiocarcinoma.

A developmental theory of environmental enrichment.

The differential brain development induced by sensory enrichment or deprivation is most apparent in rats with low brain weights. These differences are hypothesized to represent the retarded development of environment-dependent neurons in the isolated animals.

The ACE gene and Alzheimer's disease susceptibility.

A recent study suggested that the insertion (I) allele in intron 16 of the angiotensin converting enzyme gene (ACE) is associated with Alzheimer's disease (AD) risk. In our series of 239 necropsy confirmed late onset AD cases and 342 elderly non-demented controls aged >73 years, we found significantly different ACE genotype distributions in the case and control groups (p=0.007). Homozygotes for both the I and D alleles were associated with a higher risk compared to DI heterozygotes. While the APOE epsilon4 allele was strongly associated with AD risk in our series, we found no evidence for an interaction between the APOE and ACE loci. In addition, no interactions were observed between ACE and gender or age at death of the AD cases. A meta-analysis of all published reports (12 case-control series in total) suggested that both the II and ID ACE genotypes are associated with increased AD risk (odds ratio (OR) for II v DD 1.36, 95% confidence interval (CI)=1.13-1.63, OR for DI v DD 1.33, 95% CI=1.14-1.53, p=0.0002).

Genetic association of an LBP-1c/CP2/LSF gene polymorphism with late onset Alzheimer's disease.

OBJECTIVES: The only locus unequivocally associated with late onset Alzheimer's disease (AD) risk is APOE. However, this locus accounts for less than half the genetic variance. A recent study suggested that the A allele of the 3'UTR biallelic polymorphism in the LBP-1c/CP2/LSF gene was associated with reduced AD risk. Samples were diagnosed predominantly by clinical rather than pathological criteria. We have sought to replicate this finding in a series of necropsy confirmed, late onset AD cases and non-demented controls. METHODS: The 3'UTR polymorphism in the LBP-1c/CP2/LSF gene was typed in 216 necropsy confirmed AD cases and 301 non-demented controls aged >73 years. RESULTS: We found different LBP-1c/CP2/LSF allele distributions in our AD cases and controls (p=0.048); the A allele was associated with reduced AD risk. The allele and genotype frequencies observed in our cases and controls were similar to those previously reported. No significant effects emerged when the data were adjusted for age, sex, or apoE epsilon4 carrier status. CONCLUSIONS: Our data support LBP-1c/CP2/LSF as a candidate gene/risk factor for AD and provide justification for future studies to investigate the role of this gene in Alzheimer's disease.

Effects of vapocoolant spray on skin sterility prior to intravenous cannulation.

BACKGROUND: Alkane vapocoolant sprays evaporate rapidly, lower skin temperature and result in a temporary interruption in pain sensation. They reduce the pain of intravenous cannulation. However, concern exists that they may recontaminate the sterile cannulation site. AIM: To determine the effects of vapocoolant spray on skin sterility prior to cannulation. METHODS: Fifty patients from the emergency department of a large tertiary metropolitan hospital were enrolled in this study. Bacterial skin swabs were taken from the dorsum of both hands of each patient. From one hand, a swab was taken following standard chlorhexidine disinfection, and a second swab was taken following the application of vapocoolant spray. From the other hand, a swab was taken from unprepared (non-disinfected) skin, and a second swab was taken following vapocoolant application. Skin swabs were sent for microbiological culture and quantitative comparison. FINDINGS: The administration of vapocoolant after skin disinfection did not increase the bacterial colony count significantly: median 0.0 [interquartile range (IQR) 0.0] vs 0.0 (IQR 0.0) (P = 0.71). The administration of vapocoolant to the unprepared skin decreased the colony count significantly: median 33.5 (IQR 68) vs 3.0 (IQR 11) (P < 0.001). CONCLUSION: Alkane vapocoolant spray does not recontaminate the skin after disinfection, and should pose no increased risk of infection when used as an anaesthetic agent prior to intravenous cannulation following disinfection. While it does have inherent bactericidal activity, this is not sufficient for it to be used as the sole disinfectant.

Effects of urban density on carbon dioxide exchanges: Observations of dense urban, suburban and woodland areas of southern England.

Anthropogenic and biogenic controls on the surface-atmosphere exchange of CO2 are explored for three different environments. Similarities are seen between suburban and woodland sites during summer, when photosynthesis and respiration determine the diurnal pattern of the CO2 flux. In winter, emissions from human activities dominate urban and suburban fluxes; building emissions increase during cold weather, while traffic is a major component of CO2 emissions all year round. Observed CO2 fluxes reflect diurnal traffic patterns (busy throughout the day (urban); rush-hour peaks (suburban)) and vary between working days and non-working days, except at the woodland site. Suburban vegetation offsets some anthropogenic emissions, but 24-h CO2 fluxes are usually positive even during summer. Observations are compared to estimated emissions from simple models and inventories. Annual CO2 exchanges are significantly different between sites, demonstrating the impacts of increasing urban density (and decreasing vegetation fraction) on the CO2 flux to the atmosphere.

Ethanol vapour-fixation of rat lung for immunocytochemistry investigations.

Ethanol-vapour fixation of rat lung has been successfully employed in the immunocytochemical detection of the gastrin mucin antigen termed mucin 5AC without the need of additional antigen retrieval steps. This procedure gives good morphological preservation and provides all the benefits associated with the microscopic examination of inflated lung tissue. This simple fixation technique provides another option for use in immunocytochemical investigations of rodent lung and could be adapted for other species.

Comparison of the hepatic effects of nafenopin and WY-14,643 on peroxisome proliferation and cell replication in the rat and Syrian hamster.

Male Sprague-Dawley rats were fed control diet or diet containing 0.05% nafenopin (NAF) or 0.025% WY-14,643 (WY) and male Syrian hamsters were fed control diet or diet containing 0.25% NAF or 0.025% WY for periods of 1, 15, 40, and 60 weeks. Both NAF and WY produced a sustained increase in liver weight and induction of peroxisomal fatty acid beta-oxidation in the rat and Syrian hamster. Replicative DNA synthesis was studied by implanting osmotic pumps containing [3H] thymidine during weeks 0-1, 14-15, 39-40, and 59-60. Cell replication, determined either as the hepatocyte labelling index or by incorporation of radioactivity into liver whole homogenate DNA, was increased in rats given NAF and WY for 1 week. However, only WY produced a sustained increased in cell replication after 15-60 weeks. After 40 weeks, liver nodules and tumors were present in WY-treated rats, and these lesions were observed in all WY-treated and some NAF-treated rats after 60 weeks. In contrast to the rat, no marked effect on replicative DNA synthesis and no liver nodules and tumors were observed in Syrian hamsters given NAF and WY for up to 60 weeks. The rat study demonstrates that liver tumors are produced more rapidly by doses of peroxisome proliferators that produce a sustained stimulation of cell replication, whereas the hamster study suggests that species differences may exist in both peroxisome proliferator-induced cell replication and liver tumor formation.

Alzheimer disease is not associated with polymorphisms in the angiotensinogen and renin genes.

Hypertension has been implicated as a risk factor for Alzheimer disease (AD) and dementia in epidemiological studies of humans. It is thus possible that there are common genetic determinants for hypertension and AD. Epidemiological, clinical, and experimental data suggest that the renin-angiotensin-aldosterone system is a critical regulator of blood pressure. The presence of an MboI site in an RFLP in the renin gene and the Thr at the Met/Thr polymorphism at codon 235 (M235T) of the angiotensinogen gene have been reported to be associated with hypertension. These variants were studied in autopsy-confirmed AD cases and matched controls from the U.K. While no association was detected with the renin polymorphism, a weak deleterious effect was observed in cases homozygous for the angiotensinogen Thr allele. However, this association was not observed in a French cohort of clinically diagnosed AD cases and controls, suggesting that the initial observation was a type I error. Thus, these polymorphisms are unlikely to be associated with AD risk.

Manipulating the in vivo mRNA expression profile of FSH beta to resemble that of LH beta does not promote a concomitant increase in intracellular storage of follicle-stimulating hormone.

The beta-subunits of luteinizing hormone (LH beta) and follicle-stimulating hormone (FSH beta) are differentially expressed, and this may contribute to the unique expression and storage patterns of LH and FSH. Therefore, to determine if the in vivo expression profile of FSH beta could be altered to that of LH beta, a truncated ovine FSH beta (oFSH beta) gene, which would encode a mRNA lacking the putative destabilizing 3' untranslated region, was fused downstream of the ovine LH beta (oLH beta) promoter and expressed in transgenic mice. In two independent lines, line 16 and 17, we measured oFSH beta, mouse LH beta (mLHbeta) and mouse FSH beta (mFSH beta) mRNA levels: (i) after castration in males; (ii) after administering inhibin to ovariectomized mice; and (iii) during the oestrous cycle. In each experiment, the expression profile of oFSH beta mRNA mimicked mLH beta and not mFSH beta mRNA. In addition, after actinomycin D treatment of pituitary cultures, while mFSH beta mRNA did decay, there was no measurable decay of the oFSH beta mRNA transcript. These differences increased total FSH beta steady-state mRNA expression levels in male transgenics. However, there was no detectable increase in pituitary FSH by either radioimmunoassay or western blotting analysis of pituitary extracts. Subsequent analysis revealed that pituitary FSH beta in line 16 was heavily glycosylated; in contrast, pituitary FSH beta in line 17 was largely unmodified. These differences in post-translational modification of the beta-subunit, and the lack of intracellular storage, contributed to increased plasma FSH levels and ovulation rate in line 16, but not line 17. In conclusion, the expression profile of oFSH beta mRNA was manipulated to mimic mLH beta mRNA and this increased FSH beta mRNA expression levels, but did not increase storage of FSH. This suggests that, regardless of the levels of synthesis, post-translational sorting preferentially promotes FSH secretion from the pituitary.

The BACE gene: genomic structure and candidate gene study in late-onset Alzheimer's disease.

Alzheimer's disease (AD) pathology is characterized by beta-amyloid plaques and neurofibrillary tangles. Studies of autosomal dominant early-onset AD mutations suggest that beta-amyloid overproduction is sufficient to cause AD. Recently, the BACE gene, which encodes beta-secretase, the rate limiting enzyme in beta-amyloid formation, has been identified. Since this gene is a strong candidate gene for late-onset AD because of its function, we have characterized its genomic organization and identified two polymorphisms. Neither of these polymorphisms were associated with AD risk in genetic association studies comparing autopsy-confirmed late-onset AD cases and age-matched non-demented controls. Thus, we find no evidence that this locus influences risk for late-onset AD.

Blood pressure and stroke in an elderly English population.

The relation between blood pressure and subsequent stroke was examined in a four-year follow-up study of a geographically defined population sample of 2704 people aged 65 and over resident in South Tyneside in 1975. In men there was a significant relation between stroke and a history of diagnosed high blood pressure and the taking of antihypertensive medication. Stroke was not associated with either of these factors in women. Stroke incidence increased with blood pressure in men, more consistently with systolic than with diastolic pressure, but neither systolic nor diastolic pressure was related to stroke in women. In the population studied there seems little scope for the primary prevention of stroke in elderly women by detection and treatment of hypertension. Screening for the top quartile of blood pressure in men would have a sensitivity of 30% and a predictive value of 5% for stroke in the next four years. However, the extrapolation of epidemiological findings from one region or period to another may be expected to be less appropriate for the elderly than for younger people.

Secular trends in proximal femoral fracture, Oxford record linkage study area and England 1968-86.

OBJECTIVE: To study hospital admission rates for fractures of the proximal femur over a period when incidence is reported to have increased, compensating for known lack of precision in coding, excluding nonemergency admissions and transfers, and modelling for age, period, and cohort effects. DESIGN: Validation of coding of a sample of hospital admissions followed by study of two sets of routinely collected statistical abstracts of hospital records; graphical analysis and statistical modelling were used to search for period and cohort effects. SETTING: Oxfordshire and west Berkshire in 1968-86, covered by the Oxford record linkage study (ORLS), and ENGLAND in 1968-85, covered by the hospital inpatient enquiry (HIPE). The ORLS and HIPE datasets are almost independent (ORLS contributed about 1.8% of the HIPE data). SUBJECTS: Records of patients aged 65 and over. OUTCOME MEASURES: Admission rates for fractured neck of femur and fracture of other and unspecified parts of femur (N820 and N821), and evidence of period and cohort effects. RESULTS: The validation study indicated that it was important to combine the codes 820 and 821 in this age group. Admission rates increased over the period studied in both HIPE and ORLS datasets. In HIPE the pattern was of two plateaux separated by a period of rapid rise in the late 1970s. In the ORLS data there was a more steady rise. Statistical analysis showed significant period and cohort effects but much of this was attributable to the component of the model common to both period and cohort effects (termed "drift"). CONCLUSIONS: The finding that admission rates increased in both datasets, combining relevant codings and restricting analysis to emergency admissions, strongly suggests that the rise was real. At least part of the period effect in the HIPE data, however, might be attributable to a sampling artefact. The cohort effect in incidence rates of femoral fracture has not been previously shown and would be compatible with a number of aetiological hypotheses.

Studies on the induction of cholangiofibrosis by coumarin in the rat.

The histogenesis of coumarin-induced cholangiofibrosis in the rat has been determined. Proliferation of ductal structures was preceded by extensive damage to hepatocytes in the centrilobular region. Focal proliferation of ducts and fibrous tissue was present at 3 months and typical areas of cholangiofibrosis at 6 months. By 18 months the lesion was extensive and contained areas showing bizarre histological features suggestive of malignancy although no evidence of extra-hepatic metastasis was found. The lesion in animals returned to standard diet showed varying degrees of involution with extensive atrophy and fibrosis. A number of parameters of hepatic mixed function oxidase activity were reduced during the initial treatment period, at later times there was recovery of some microsomal enzyme activities. The activity of gamma-glutamyltransferase and the hepatic content of non-protein sulphydryl groups, in contrast, were raised throughout the treatment period.

Effect of prolonged administration of clofibric acid and di-(2-ethylhexyl)phthalate on hepatic enzyme activities and lipid peroxidation in the rat.

Male Sprague-Dawley rats were fed diets containing either 0.5% clofibric acid (CA) or 2% di-(2-ethylhexyl)phthalate (DEHP) for 2 years. Both compounds produced liver enlargement which was accompanied by the formation of liver nodules. Hepatic peroxisomal and microsomal fatty acid oxidising enzyme activities were induced in both large nodules and host tissue (i.e. tissue remaining after removal of large nodules) preparations from CA and DEHP treated rats. In contrast, little change in catalase activity was observed and the activities of cytosolic GSH peroxidase and GSH S-transferases were markedly reduced. Increased lipid peroxidation was observed by measurement of conjugated dienes in host tissue homogenates from CA and DEHP treated rats. Microsomal NADPH-dependent lipid peroxidation was also stimulated. Histological examination revealed extensive lipofuscin deposition in non-nodular, but not in nodular, tissue sections from treated rats. These results demonstrate that prolonged peroxisome proliferation can result in lipid peroxidation and that certain enzymes which metabolise hydrogen peroxide and organic hydroperoxides are either little affected or markedly inhibited.

Sustained induction of hepatic xenobiotic metabolising enzyme activities by phenobarbitone in C3H/He mice: relevance to nodule formation.

Phenobarbitone (PB) was administered to male C3H/He mice at a dose of 85 mg/kg/day in a semisynthetic diet for up to 90 weeks. Throughout the treatment period a sustained induction of a number of parameters of hepatic Phase I and Phase II xenobiotic metabolism was observed. Histological examination revealed hypertrophy of the centrilobular cells of the liver lobule in PB treated mice and after 25 weeks small basophilic nodules were found in control and PB treated animals. In addition eosinophilic nodules, which were often large, developed in PB treated mice. Xenobiotic metabolising enzyme activities in large excised nodules after 70 or 90 weeks of PB treatment were either similar to or greater than those present in surrounding host tissue. Both phenobarbitone- and polycyclic hydrocarbon-type mixed function oxidase enzyme activities were induced in large nodules. In conclusion, PB produced a sustained induction of xenobiotic metabolising enzymes both in host tissue and in large eosinophilic nodules. The formation of these nodules in C3H/He mice was thus not associated with any failure of induction of hepatic xenobiotic metabolism.

The Hypertension in the Very Elderly Trial (HYVET). Rationale, methodology and comparison with previous trials.

The Hypertension in the Very Elderly Trial (HYVET) is a multicentre, open, randomised, controlled trial. The aim of this trial is to investigate the effect of active treatment on stroke incidence in hypertensive patients over the age of 80 years. Secondary end-points include total cardiovascular mortality and morbidity. Entry criteria include a sustained sitting systolic blood pressure of 160 to 219mm Hg plus a sustained sitting diastolic pressure of 95 to 109mm Hg. Also required is a standing systolic blood pressure of at least 140mm Hg. Patients must give their informed consent, and be free of congestive heart failure requiring treatment, gout, renal failure or a recent cerebral haemorrhage. Patients are to be randomised to 3 groups-(i) no treatment; (ii) treatment with a diuretic [bendroflumethiazide (bendrofluazide)]; or (iii) treatment with an angiotensin converting enzyme (ACE) inhibitor (lisinopril). Starting dosage for bendroflumethiazide and lisinopril is 2.5 mg/day. In order to achieve goal sitting systolic and diastolic blood pressures (< 150/80 mm Hg), a doubling of the dosage is allowed. Furthermore, slow release diltiazem (120 mg/day increasing to 240 mg/day if required) may be added to the medication of the actively treated groups. These drugs have been chosen as inexpensive and appropriate representatives of their therapeutic classes. 700 patients in each group (a total of 2100) will be sufficient to detect a 40% difference in cerebrovascular events between no treatment and active treatment (alpha = 0.01, 1-beta = 0.90). These numbers will also detect a difference in total mortality of 25% and in cardiovascular mortality of 35%. The pilot phase of the trial has been started with support from the British Heart Foundation. Centres which are interested in taking part should contact C.J. Bulpitt or any of the other authors.

Ageing in the South Pacific. Physical changes with urbanization.

The process of ageing, the place the elderly hold in the South Pacific societies and the care they receive as they move from adult independence to geriatric dependence varies considerably in different Pacific Polynesian populations. This provides unusual opportunity to examine the physical changes of ageing in people of the same broad genetic make-up exposed to environmental changes brought about by urbanization. Epidemiological surveys carried out since 1962 among New Zealand Maoris, Tongans, Cook Island Maoris in Rarotonga and Pukapuka, and Tokelauans living in Tokelau and following migration to New Zealand, provide the main data base for this presentation. The pattern of blood pressure, body weight, serum lipids and clinical disorders show considerable variation which relate most closely to the adoption of westernized life-style and moving into an urban environment. Analysis of the ECG pattern, in Pukapukans, in whom blood pressure shows only a minor increase with age, compared with age and sex-matched subjects studied in Newcastle, England provide insights into the ageing heart. An examination of mortality based on risk factors at entry shows an inverse relationship of serum cholesterol to total mortality in New Zealand Maori men and women, in Tokelau men but not women. Increasing systolic blood pressure was related to mortality in New Zealand Maori men, Tokalau men and Caucasian women, but not in the other race sex groups. The pattern of ageing and risk factors must clearly be examined in individual populations because while death is the end the pathways vary.

Tumours and malformations in the adult offspring of cyclophosphamide-treated and control male rats--preliminary communication.

Adult offspring aged 52-104 weeks, from male Sprague-Dawley rats treated chronically with cyclophosphamide (CP) were examined for tumours and gross abnormalities. Litter size at birth and at weaning was found to be greatly reduced as a result of paternal CP treatment. No unusual abnormalities were found at post-mortem examination but there was an increase in the incidence of hydronephrosis in offspring from CP-treated males compared with offspring from control males. This increase could have been indirectly caused by CP-treatment through reduced litter size. Histological examination of 26 tumours showed a variety of tumour types in the offspring of CP-treated and control males. Two of the four uterine tumours in offspring from CP-treated males were examined histologically; one was a sarcoma and the other an adenocarcinoma. Although no uterine tumours were found in offspring from control males, it is not clear whether this difference in frequency was treatment-related. The most common tumour site in female offspring from both CP-treated and control males was the mammary gland, and all six of these tumours which were examined histologically were adenofibromas. Abnormal karyotypes were observed in 2 out of 21 offspring showing abnormalities from CP-treated males and none out of 2 offspring with abnormalities from control males. These were not associated with tumours. It was concluded from this limited study that there was no clear evidence of increased tumour incidence in the offspring from CP-treated males. There was an indication that abnormal karyotypes may have been caused by the paternal CP treatment and these abnormalities persisted into adulthood.