Caveolin-1 deficiency protects against mesangial matrix expansion in a mouse model of type 1 diabetic nephropathy.

AIMS/HYPOTHESIS: Glomerular matrix protein accumulation, mediated largely by resident mesangial cells (MCs), is central to the pathogenesis of diabetic nephropathy. We previously showed that caveolin (CAV)-1/caveolae mediate matrix upregulation by MCs in response to high glucose and TGFß, two important pathogenic mediators of diabetic glomerular sclerosis. Here, we evaluated the in vivo role of CAV-1/caveolae in the development of diabetic nephropathy. METHODS: Diabetes was induced in Cav1-knockout (KO) mice and their wild-type (WT) counterparts by streptozotocin injection. After 10 months, kidneys were evaluated for the development of nephropathy, including glomerular sclerosis and upregulation of matrix proteins. Parallel experiments assessing glucose-induced matrix upregulation were carried out in MCs isolated from KO mice. RESULTS: KO diabetic mice developed hyperglycaemia and renal hypertrophy, but were protected from developing albuminuria and glomerular sclerosis compared with WT mice. KO mice were significantly protected from the upregulation of glomerular collagen I, fibronectin, connective tissue growth factor (CTGF) and TGFß. In vitro, glucose induced collagen I A1 promoter activation and collagen I, fibronectin and CTGF protein upregulation in WT but not KO MCs. Re-expression of Cav1 in KO cells restored this response. CONCLUSIONS/INTERPRETATION: Cav1 deletion rendered significant protection from glomerular matrix accumulation and albuminuria in a mouse model of type 1 diabetes. These studies provide a foundation for the development of renal-targeted interference with CAV-1/caveolae as a novel approach to the treatment of diabetic nephropathy.

High glucose-induced RhoA activation requires caveolae and PKCß1-mediated ROS generation.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We previously showed that RhoA activation by high glucose in mesangial cells (MC) leads to matrix upregulation (Peng F, Wu D, Gao B, Ingram AJ, Zhang B, Chorneyko K, McKenzie R, Krepinsky JC. Diabetes 57: 1683-1692, 2008). Here, we study the mechanism whereby RhoA is activated. In primary rat MC, RhoA activation required glucose entry and metabolism. Broad PKC inhibitors (PMA, bisindolylmaleimide, Gö6976), as well as specific PKCß blockade with an inhibitor and small interfering RNA (siRNA), prevented RhoA activation by glucose. PKCß inhibition also abrogated reactive oxygen species (ROS) generation by glucose. The ROS scavenger N-acetylcysteine (NAC) or NADPH oxidase inhibitors apocynin and DPI prevented glucose-induced RhoA activation. RhoA and some PKC isoforms localize to caveolae. Chemical disruption of these microdomains prevented RhoA and PKCß1 activation by glucose. In caveolin-1 knockout cells, glucose did not induce RhoA and PKCß1 activation; these responses were rescued by caveolin-1 reexpression. Furthermore, glucose-induced ROS generation was significantly attenuated by chemical disruption of caveolae and in knockout cells. Downstream of RhoA signaling, activator protein-1 (AP-1) activation was also inhibited by disrupting caveolae, was absent in caveolin-1 knockout MC and rescued by caveolin-1 reexpression. Finally, transforming growth factor (TGF)-ß1 upregulation, mediated by AP-1, was prevented by RhoA signaling inhibition and by disruption or absence of caveolae. In conclusion, RhoA activation by glucose is dependent on PKCß1-induced ROS generation, most likely through NADPH oxidase. The activation of PKCß1 and its downstream effects, including upregulation of TGF-ß1, requires caveolae. These microdomains are thus important mediators of the profibrogenic process associated with diabetic nephropathy.

HB-EGF release mediates glucose-induced activation of the epidermal growth factor receptor in mesangial cells.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We showed that transactivation of the epidermal growth factor receptor (EGFR) is an important mediator of matrix upregulation in mesangial cells (MC) in response to high glucose (HG). Here, we study the mechanism of EGFR transactivation. In primary MC, EGFR transactivation by 1 h of HG (30 mM) was unaffected by inhibitors of protein kinase C, reactive oxygen species, or the angiotensin II AT1 receptor. However, general metalloprotease inhibition, as well as specific inhibitors of heparin-binding EGF-like growth factor (HB-EGF), prevented both EGFR and downstream Akt activation. HB-EGF was released into the medium by 30 min of HG, and this depended on metalloprotease activity. One of the metalloproteases shown to cleave proHB-EGF is ADAM17 (TACE). HG, but not an osmotic control, activated ADAM17, and its inhibition prevented EGFR and Akt activation and HB-EGF release into the medium. siRNA to either ADAM17 or HB-EGF prevented HG-induced EGFR transactivation. We previously showed that EGFR/Akt signaling increases transforming growth factor (TGF)-ß1 transcription through the transcription factor activator protein (AP)-1. HG-induced AP-1 activation, as assessed by EMSA, was abrogated by inhibitors of metalloproteases, HB-EGF and ADAM17. HB-EGF and ADAM17 siRNA also prevented AP-1 activation. Finally, these inhibitors and siRNA prevented TGF-ß1 upregulation by HG. Thus, HG-induced EGFR transactivation in MC is mediated by the release of HB-EGF, which requires activity of the metalloprotease ADAM17. The mechanism of ADAM17 activation awaits identification. Targeting upstream mediators of EGFR transactivation including HB-EGF or ADAM17 provides novel therapeutic targets for the treatment of diabetic nephropathy.

EGFR-PLCgamma1 signaling mediates high glucose-induced PKCbeta1-Akt activation and collagen I upregulation in mesangial cells.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We have recently shown that epidermal growth factor receptor (EGFR) transactivation mediates high glucose (HG)-induced collagen I upregulation through PI3K-PKCbeta1-Akt signaling in mesangial cells (MC). Phospholipase Cgamma1 (PLCgamma1) interacts with activated growth factor receptors and activates classic PKC isoforms. We thus studied its role in HG-induced collagen I upregulation in MC. Primary rat MC were treated with HG (30 mM) or mannitol as osmotic control. Protein kinase activation was assessed by Western blotting and collagen I upregulation by Northern blotting. Diabetes was induced in rats by streptozotocin. HG treatment for 1 h led to PLCgamma1 membrane translocation and Y783 phosphorylation, both indicative of its activation. Mannitol was without effect. PLCgamma1 Y783 phosphorylation was also seen in cortex and glomeruli of diabetic rats. HG induced a physical association between EGFR and PLCgamma1 as identified by coimmunoprecipitation. PLCgamma1 activation required EGFR kinase activity since it was prevented by the EGFR inhibitor AG1478 or overexpression of kinase-inactive EGFR (K721A). Phosphoinositide-3-OH kinase inhibition also prevented PLCgamma1 activation. HG-induced Akt S473 phosphorylation, effected by PKCbeta1, was inhibited by the PLCgamma inhibitor U73122. PLCgamma1 inhibition or downregulation by small interference RNA also prevented HG-induced collagen I upregulation. Our results indicate that EGFR-PLCgamma1 signaling mediates HG-induced PKCbeta1-Akt activation and subsequent collagen I upregulation in MC. Inhibition of EGFR or PLCgamma1 may provide attractive therapeutic targets for the treatment of diabetic nephropathy.

Early changes produced in mouse skin by the application of three middle distillates.

It has been reported by the American Petroleum Institute (API) that dermal applications of certain middle distillates of mineral oils can result in high incidences of skin tumours in mice. This was unexpected as the polycyclic aromatic hydrocarbon (PAH) levels in these were below detection limits. To examine the possible role of tissue injury in the induction of tumours, the skin reactions produced by thrice weekly applications of three middle distillates similar to those tested by the API were examined grossly and histopathologically at intervals up to 6 weeks. Various reference materials and oils were used as controls. Preliminary histological examination showed that severe skin damage was present from week 1 onwards in mice treated with the three middle distillates, two of them producing epidermal loss and ulceration. Marked epidermal hyperplasia was produced by all three middle distillates. These findings support the view that regenerative epidermal hyperplasia due to repeated severe skin damage may have exerted a powerful promotional effect in the production of the skin tumours by middle distillates in the API study.

Prolonged sulfonylurea-induced hypoglycemia in diabetic patients with end-stage renal disease.

Renal impairment is a recognized risk factor for prolonged hypoglycemia, but predisposing characteristics in patients with advanced renal impairment have not been studied. We observed prolonged hypoglycemia in a number of patients with end-stage renal disease (ESRD) and conducted a case-control study at two Canadian centers to identify such risk factors. Through hospital, pharmacy, and dialysis program records, we retrospectively identified 7 case patients and 31 controls with ESRD and type 2 diabetes using oral hypoglycemic monotherapy. Control patients had no history of hospital admission for prolonged hypoglycemia. All case patients and 28 controls were receiving glyburide (glibenclamide in Europe); the remainder were treated with tolbutamide. Duration of intravenous treatment for hypoglycemia ranged from 28 to 256 hours, with 83 g to 2 kg of glucose administered per episode. Preceding treatment with glyburide varied from 2 days to 13 years. Univariate analyses showed a recent decline in oral intake (odds ratio [OR], 81; 95% confidence interval [CI], 3.6 to 1,840), previous hypoglycemic episodes (OR, 15; 95% CI, 0.77 to 297), longer duration of diabetes (22 versus 12 years; P = 0.008), and a history of cerebrovascular disease (OR, 7. 0; 95% CI, 1.0 to 47) to be associated with prolonged hypoglycemia. No association between prolonged hypoglycemia and age, sex, beta blockers, angiotensin-converting enzyme inhibitors, oral hypoglycemic dose, or duration of treatment was identified. This study describes the potentially devastating effect of sulfonylurea-based oral hypoglycemic therapy in ESRD. Patients at greatest risk appear to be those with reduced intake, previous hypoglycemic episodes, and longer duration of diabetes. We describe the mechanisms for observed hypoglycemia and suggest that alternative drugs may be considered in this patient group.

Interaction of benzo [A] pyrene and a hyperplastic agent in epidermal nuclear enlargement in the mouse. A dose response study.

Experiments were conducted on the effects of various dose levels of benzo [a]pyrene (BP) on nuclear size in mouse interfollicular epidermis over a 3-day period. Topical application of BP was made with or without croton oil (CO) (0.1 or 0.5%) in the vehicles acetone, toluene and methyl ethyl ketone (MEK). Nuclear size was measured on histological sections either manually or by Quantimet Image Analyser. Vehicle controls treated with 0.1 or 0.5% CO in acetone or MEK gave rise to epidermal hyperplasia with some nuclear enlargement and toluene without CO produced a similar response. It was found that when BP was applied in a vehicle capable of inducing hyperplasia, the nuclear enlargement produced was greater than that produced by either the vehicle control or BP in a non-irritant vehicle. The enhancement of response to BP when tested in the presence of a hyperplastic agent resulted in lower concentrations of BP being detectable. As the levels of BP detectable by nuclear enlargement under these conditions compared reasonably well with those detectable in long-term tests, this system might be usable as a basis for a short-term test for carcinogens.

Evidence for and possible mechanisms of non-genotoxic carcinogenesis in mouse skin.

Most chemicals that produce skin cancer are genotoxic by in vitro and in vivo short-term assays and produce a high incidence of skin cancer within a year if optimal doses are applied. If in long-term skin painting studies one or two tumours in 50 mice are observed there is a general consensus that no carcinogenic activity can be claimed and it has been suggested that if up to 10% tumours are induced by irritant substances this could be due to an enhancement of spontaneous tumour incidence. Observations of skin tumour incidences higher than 10% with non-genotoxic substances, usually after a long latent period, is considered to represent evidence for a non-genotoxic mechanism. Examples of such substances include croton oil, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), sodium hydroxide, potassium hydroxide, phenol, dodecylbenzene and petroleum-derived middle distillates. Two distinct mechanisms appear to be involved in the production of tumours by a non-genotoxic substance. The first of these is that seen with the strong promoting agents. These, by binding to and activating protein kinase C, appear to directly stimulate sustained epidermal hyperplasia without severe skin damage. The other appears to involve substances producing severe skin damage either by a direct caustic effect or by cumulative irritancy. These changes give rise to marked epidermal hyperplasia with repeated episodes of regeneration and damage. The tumour induction by both mechanisms probably results from oncogene activation and it is possible that oxidative enzymes from inflammatory cells may be involved in the activation process. Various reasons are given why non-genotoxic carcinogenesis in the skin is considered not to be relevant to man and ways of recognising and avoiding its occurrence in animals studies are recommended.

Utility of ultrasonographic venous assessment prior to forearm arteriovenous fistula creation.

AIM: The purpose of this study was to evaluate the clinical utility of Doppler ultrasound (US) prior to native forearm arteriovenous fistula (AVF) creation. MATERIALS AND METHODS: US mapping was carried out pre-operatively to evaluate the major veins and arteries in the appropriate arm. One hundred and 6 patients were identified retrospectively over 2 years with complete clinical and US data. A failed fistula was defined as an inability to provide blood flow to meet adequacy targets by 6 months (urea reduction ratio > or = 65%). RESULTS: Twenty-nine patients (27.4%) had successful forearm AVFs. The mean minimum forearm cephalic vein diameter (CVD) was 2.51 +/- 0.14 and 2.23 +/- 0.06 mm in successful and failed fistulae, respectively (p = 0.04). This result was primarily due to differences observed in women. A receiver operator curve analysis showed that a cutpoint of 2.6 mm for minimum forearm CVD had the greatest predictive value with a likelihood ratio of 3.94 (95% CI: 1.97 - 7.84) for fistula failure. Multivariate logistic regression analysis determined that male gender and minimum forearm CVD were the only significant predictors for fistula success with odds ratios of 3.90 (95% CI: 1.30 - 11.68) and 2.31 (95% CI: 1.00 - 5.43), respectively. The study is limited by the possibility that US results in patients may have lead to an alternative type of access being attempted. CONCLUSIONS: US mapping prior to forearm AVF creation is of modest benefit. Only male gender and minimum forearm CVD were predictive of AVF success.

A three-generation reproduction study of Ponceau 4R in the rat.

Ponceau 4R was fed to three generations of rats, at dietary concentrations to provide 0, 50, 500 or 1250 mg/kg body weight/day. In each generation treated groups consisted of 36 rats of each sex while 60 females served as controls. Apart from the F0 generation, which started treatment as weanlings, treatment was continued throughout the study, providing in utero exposure of all offspring. The F0 generation was bred twice, on the first occasion to provide animals for the next generation and for a long-term study, and a second time to provide data on in utero and post-partum development. In each generation approximately one third of the females from each group were killed before parturition to provide data on in utero development. The foetuses from these animals were examined for skeletal abnormalities. Remaining animals were allowed to litter and the offspring were monitored for 21 days after birth for survival and development. All animals were killed and subjected to a post-mortem examination which, for a proportion of each group in each generation, included recording of organ weights. Although a few adult rats died during the study these deaths were not associated with treatment. Fur of the treated animals was coloured pink, and faeces and caecal contents of animals from the two highest dose groups were yellow, the faeces also being softer than those of the controls. Treatment had no observed effect on clinical observations, body weight or food and water intake at any stage of the study. Animals fed the two highest doses for prolonged periods had enlarged caeca, but this effect was not seen in weanling animals on the same treatment. Neither the caecal enlargement nor the liver weights seen in the F2 and F3 offspring were considered to be an adverse effect of treatment. No treatment-related effects were seen in the uterine contents of females at any generation, but the skeletons of treated foetuses showed a slightly more advanced development than those of the controls. Postnatal development of offspring was not affected by treatment at any stage of the study. Tissues from the F3 animals were examined by light microscopy and revealed no treatment-related effects. It is concluded that the no-adverse-effect level for Ponceau 4R is 1250 mg/kg body weight/day.

Short-term toxicity study of metatartaric acid in rats.

Groups of rats were given metatartaric acid in the drinking-water in concentrations of 0 (control), 0.1, 0.5 or 3.0% for 18 wk. No effects associated with treatment were seen in the results of the haematological examinations and serum analyses. The treated animals consumed less water and food than the controls, probably because of the unpalatability of the test material. Administration of the 3% solution was associated in males with a reduced growth rate, some impairment of urine-concentrating ability during prolonged water deprivation (also seen in males on 0.5%) and histopathological changes in the stomach indicative of an inflammatory response in the submucosal layer. Both sexes of the 3% group showed an increase in relative kidney weight, without accompanying histopathological change. The no-untoward-effect level in this study was 0.1% metatartaric acid in the drinking-water, equivalent to a mean daily intake of 80 mg/kg body weight in the males and 130 mg/kg in the females.

Homocysteine induces mesangial cell apoptosis via activation of p38-mitogen-activated protein kinase.

Hyperhomocysteinemia is prevalent among patients with chronic kidney disease (CKD) and has been linked to progressive kidney and vascular diseases. Increased glomerular mesangial cell (MC) turnover, including proliferation and apoptosis, is a hallmark of CKD. Activation of p38-mitogen-activated protein kinase (p38-MAPK) has been linked to apoptosis in many cell lines. Accordingly, we studied the effect of homocysteine (Hcy) on MC p38-MAPK signalling and apoptosis. Hcy (50 microM/24 h) increased MC apoptosis as determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) and single-stranded DNA (ssDNA) analysis. In addition to increases in pro-caspase-3 protein and caspase-3 activity, cells exposed to Hcy manifested enhanced reactive oxygen species content. Hcy increased p38-MAPK activity (fivefold), with maximal effect at 50 microM and 20 min; p38-MAPK activation was attenuated by N-acetylcysteine (Nac) and catalase (Cat), further indicating that the effect was via oxidative stress. Confocal microscopy revealed activation and nuclear translocation of p38-MAPK that was attenuated by Cat. In addition, Hcy-induced apoptosis as determined by TUNEL and ssDNA assay was abrogated by Nac, Cat, and SB203580 (p38-MAPK inhibitor). We conclude that in MC, Hcy (i) activates p38-MAPK and increases p38MAPK nuclear translocation via an oxidative stress dependent mechanism and (ii) induces DNA damage and apoptosis that is dependent on oxidative stress and p38-MAPK activation.

Collagen I induction by high glucose levels is mediated by epidermal growth factor receptor and phosphoinositide 3-kinase/Akt signalling in mesangial cells.

AIMS/HYPOTHESIS: Glomerular matrix accumulation is a hallmark of diabetic nephropathy. Recent data have linked the serine/threonine kinase protein kinase B (Akt) to matrix modulation. Here, we studied its role in high glucose-induced collagen elaboration by mesangial cells. METHODS: Primary rat mesangial cells were treated with high glucose levels (30 mmol/l) or mannitol as osmotic control. Western blots, northern blots, ELISA and immunohistochemistry were used for assessment. Diabetes was induced in rats by streptozotocin. RESULTS: Phosphorylated Akt at S473 (pAktS473), corresponding to Akt activation, was seen in diabetic glomeruli. In mesangial cells, high glucose levels induced pAktS473 by 20 min. This was sustained to 72 h, while mannitol had no effect. Akt activation by kinase assay and phosphorylation on threonine 308 was also observed. Phosphoinositide 3-kinase (PI3K) inhibitors LY294002 (20 micromol/l) and wortmannin (100 nmol/l) prevented pAktS473. Collagen IA1 transcript and collagen I protein upregulation by high glucose levels were inhibited by PI3K blockade, as was collagen I secretion into the medium (ELISA). Dominant-negative Akt overexpression also inhibited high glucose-induced collagen IA1 transcript and collagen I protein production. Since signalling through the epidermal growth factor receptor (EGFR) can activate PI3K-Akt, we studied its activation by high glucose levels. EGFR was correspondingly activated by 10 min; mannitol had no effect. EGFR activation was also seen in glomeruli from diabetic rats and co-localised with collagen IA1 in diabetic glomeruli. Specific EGFR inhibition (AG1478, 5 micromol/l or dominant-negative EGFR) blocked high glucose-induced pAktS473, phosphorylation on threonine 308 and activation of the EGFR downstream target p44 extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase. Finally, EGFR inhibition also blocked high glucose-induced collagen I upregulation at transcriptional and protein levels. CONCLUSIONS/INTERPRETATION: We conclude that EGFR-PI3K-Akt signalling mediates high glucose-induced collagen I upregulation in mesangial cells and that this pathway is activated in diabetic glomeruli. Targeting its components may provide a new therapeutic approach to diabetic kidney disease.

Review of chemical and UV light-induced melanomas in experimental animals in relation to human melanoma incidence.

In a few epidemiological studies on oil refinery workers, a slight excess of melanoma incidence has been reported. To see if this might be linked to exposure to polycyclic aromatic hydrocarbons (PAHs) contained in refinery streams, a review of animal data on the relationship between PAH exposure, UV light and melanoma induction has been carried out and compared with human data. This revealed that the highly carcinogenic PAH 7,12-dimethylbenz[a]anthracene (DMBA) was capable of inducing melanomas in hamsters, mice and guinea pigs, but only under certain experimental conditions. Evidence suggested that other carcinogenic PAHs were unable to induce melanomas. As high dose levels of DMBA were generally required to produce melanomas, it was not considered that the amounts present in refinery streams would be sufficient to account for an increase in melanoma incidence in exposed workers. This conclusion was substantiated by the failure of petroleum-derived complex hydrocarbon mixtures to produce melanomas in animals or man and by drawing attention to the absence of any association between melanoma incidence and the incidence of other skin cancers in man. If PAHs were responsible for an increase in melanoma incidence, an increase in other skin tumours would also be expected. It was concluded that animal data, taken in conjunction with other information, do not suggest that PAH exposure is likely to be the cause of any elevation in melanoma incidence in refinery workers. More detailed epidemiological findings would be required to establish whether any excess incidence of melanomas was due to sunlight, other risk factors or chance occurrences.

Carcinogenic oil fractionation and nuclear enlargement in mouse skin.

A non-solvent-refined oil, previously shown to produce tumours in a long-term skin painting assay and a positive result in the mouse skin nuclear enlargement test, was separated using silica gel elution techniques into eight fractions. Fraction 1, which contained saturated and 1-2 ring aromatic hydrocarbons, was used to dilute the other fractions to produce solutions equivalent to and twice their original concentration in the intact oil. Fraction 1, diluted fractions 2-8, the reconstituted oil, the original oil and a negative control oil were examined for their ability to produce nuclear enlargement. The degree of nuclear enlargement observed with fraction 1 was considered to indicate marginal or no carcinogenic activity. With the fraction that contained 2-3 ring aromatic compounds, weak carcinogenic activity could not be ruled out but it is unlikely that these components played a major role in the carcinogenicity of the oil. The greatest activity was identified in the fraction containing the total 3/4-6 ring polycyclic aromatic hydrocarbons (PAHs) and further fractionation of this suggested that the parent 4-6 ring PAHs were responsible for more than half the activity of the intact oil, despite being present at only 0.06%. The fraction that contained polar compounds showed no activity. The reconstituted oil showed slightly less activity than the original oil. Other fractions, which contained alkylated PAHs, did not seem to have much activity. These findings, which are in close agreement with published evidence on tumour induction by fractions of oils and coal liquids, suggest that the components responsible for inducing nuclear enlargement are the same as those responsible for skin tumour induction.(ABSTRACT TRUNCATED AT 250 WORDS)

The lethal and hepatocarcinogenic effects of dimethylnitrosamine injection in the newt Triturus helveticus.

Single intraperitoneal injections of dimethylnitrosamine (DMN) in concentrations of up to 16 mg/g body weight, failed to have any lethal effect in newts. This treatment also failed to induce tumours in newts maintained for one year after injection.Six or 7 injections of 16 mg/g body weight of DMN, administered over a period of 3-4 weeks, gave rise to a short-term lethal effect due to liver necrosis and in the long term to liver tumours. The tumours induced were of similar type to nitrosamineinduced tumours in rats, one being an anaplastic liver tumour and the other nodular hepatic cell tumours.Attempts to maintain the tumours by grafting failed, probably due to homograft rejection.

Stress-responsive signal transduction mechanisms in glomerular cells.

Mechanical stresses appear to play a key role in the progression of glomerular diseases that are characterized by increased transcapillary hydraulic pressure. Glomerular mesangial cells proliferate and produce extracellular matrix proteins in vivo in such diseases. Mesangial cell responses to pulsatile mechanical stimuli have been studied extensively in vitro during the past few years. Mechanical signals are sensed at the cell membrane and propagated through the cytoplasm, and result in the activation of transcription factors that elicit production of prosclerotic cytokines and matrix proteins, and cell proliferation. Endothelial cells are exposed to shear and pulsatile stress and show some similar responses in other vascular beds.

The dermal bioavailability of radiolabelled benzo[a]pyrene from acetone or from oils of differing viscosity, assessed by DNA and protein binding.

Tritium-labelled benzo[a]pyrene ([3H]BaP) was applied to mouse skin in acetone or mineral oils of differing viscosity. Epidermal DNA and protein were extracted after 24 or 48 h and the degree of adduct formation determined by the radioactivity present. When [3H]BaP was applied in acetone, the degree of DNA and protein binding was around 15-20 times greater than that observed when a low-viscosity oil was used as a vehicle. When applied in oils of differing viscosity, however, only a twofold difference was seen across the whole viscosity range (13.5 cSt* at 40 degrees C to 1665 cSt at 60 degrees C). From measurements made of urine and faecal radioactivity and from small-scale investigations using other routes of administration, it was clear that the grooming activity of the animals had a marked effect on skin absorption and macromolecular binding. It is possible that greater grooming activity with low-viscosity oils may explain why oil viscosity did not have a greater effect on binding levels, but further studies are needed to investigate this. These findings may have important implications in the interpretation of long-term skin painting studies and may assist in the interpretation of analytical data and short-term biological assays.

Patch testing in the rabbit using a modified human patch test method. Application of histological and visual assessement.

A 5 h, semi-occlusive human patch test repeated daily for 5 days, was modified for use in the rabbit Using this method, the relative irritancy of three cosmetic samples of known human irritancy was determined in the rabbit. In addition, attention was paid to assessment methods and a detailed histological scoring system is described as well as two methods of visual assessment. In order to compare the effectiveness of these methods, the reaction to a series of concentrations of sodium lauryl sulphate was examined. All three assessment methods gave similar results and the merits of combining histological and visual assessment are discussed.