Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension.

BACKGROUND: Epigenetic regulation of vascular remodeling in pulmonary hypertension (PH) is poorly understood. Transcription regulating, histone acetylation code alters chromatin accessibility to promote transcriptional activation. Our goal was to identify upstream mechanisms that disrupt epigenetic equilibrium in PH. METHODS: Human pulmonary artery smooth muscle cells (PASMCs), human idiopathic pulmonary arterial hypertension (iPAH):human PASMCs, iPAH lung tissue, failed donor lung tissue, human pulmonary microvascular endothelial cells, iPAH:PASMC and non-iPAH:PASMC RNA-seq databases, NanoString nCounter, and cleavage under targets and release using nuclease were utilized to investigate histone acetylation, hyperacetylation targets, protein and gene expression, sphingolipid activation, cell proliferation, and gene target identification. SPHK2 (sphingosine kinase 2) knockout was compared with control C57BL/6NJ mice after 3 weeks of hypoxia and assessed for indices of PH. RESULTS: We identified that Human PASMCs are vulnerable to the transcription-promoting epigenetic mediator histone acetylation resulting in alterations in transcription machinery and confirmed its pathological existence in PH:PASMC cells. We report that SPHK2 is elevated as much as 20-fold in iPAH lung tissue and is elevated in iPAH:PASMC cells. During PH pathogenesis, nuclear SPHK2 activates nuclear bioactive lipid S1P (sphingosine 1-phosphate) catalyzing enzyme and mediates transcription regulating histone H3K9 acetylation (acetyl histone H3 lysine 9 [Ac-H3K9]) through EMAP (endothelial monocyte activating polypeptide) II. In iPAH lungs, we identified a 4-fold elevation of the reversible epigenetic transcription modulator Ac-H3K9:H3 ratio. Loss of SPHK2 inhibited hypoxic-induced PH and Ac-H3K9 in mice. We discovered that pulmonary vascular endothelial cells are a priming factor of the EMAP II/SPHK2/S1P axis that alters the acetylome with a specificity for PASMC, through hyperacetylation of histone H3K9. Using cleavage under targets and release using nuclease, we further show that EMAP II-mediated SPHK2 has the potential to modify the local transcription machinery of pluripotency factor KLF4 (Krüppel-like factor 4) by hyperacetylating KLF4 Cis-regulatory elements while deletion and targeted inhibition of SPHK2 rescues transcription altering Ac-H3K9. CONCLUSIONS: SPHK2 expression and its activation of the reversible histone H3K9 acetylation in human pulmonary artery smooth muscle cell represent new therapeutic targets that could mitigate PH vascular remodeling.

Medical ethics: knowledge, attitude and practice among doctors in three teaching hospitals in Sri Lanka.

BACKGROUND: Medical ethics deals with the ethical obligations of doctors to their patients, colleagues and society. The annual reports of Sri Lanka Medical Council indicate that the number of complaints against doctors has increased over the years. We aimed to assess the level of knowledge, attitude and practice regarding medical ethics among doctors in three teaching hospitals in Sri Lanka. METHODS: A hospital-based cross-sectional study was conducted among doctors (n = 313) using a pre-tested self-administered, anonymous questionnaire. Chi Squared test, and ANOVA test were used to identify the significance of association between level of knowledge and selected factors. RESULTS: Most doctors (81.2%) had a poor level of knowledge on medical ethics, with postgraduate trainees showing significantly (p = 0.023, Chi square) higher level of knowledge. The average knowledge on medical ethics among doctors was significantly different between the three hospitals (p = 0.008, ANOVA). Over 95% had a favourable attitude towards gaining knowledge and advocated the need for training. The majority (69.3%) indicated awareness of unethical practices. 24.6% of respondents stated that they get a chaperone 'sometimes' during patient examination while 3.5% never do. The majority (54%) responded that they never accept gifts from pharmaceutical companies in recognition of their prescribing pattern. 12-41% of doctors participated in the study acknowledged that they 'sometime' engaged in unethical practices related to prescribing drugs, accepting gifts from pharmaceutical companies and when obtaining leave. CONCLUSION: Most doctors had a poor level of knowledge of medical ethics. Postgraduate trainees had a higher level of knowledge than other doctors. The majority showed a favourable attitude towards gaining knowledge and the need of training. Regular in-service training on medical ethics for doctors would help to improve their knowledge on medical ethics, as well as attitudes and ethical conduct.

Lipid droplet remodelling and reduced muscle ceramides following sprint interval and moderate-intensity continuous exercise training in obese males.

BACKGROUND/OBJECTIVES: In obesity, improved muscle insulin sensitivity following exercise training has been linked to the lowering of diacylglycerol (DAG) and ceramide concentrations. Little is known, however, about how improved insulin action with exercise training in obese individuals relates to lipid droplet (LD) adaptations in skeletal muscle. In this study we investigated the hypothesis that short-term sprint interval training (SIT) and moderate-intensity continuous training (MICT) in obese individuals would increase perilipin (PLIN) expression, increase the proportion of LDs in contact with mitochondria and reduce muscle concentrations of DAGs and ceramides. METHODS: Sixteen sedentary obese males performed 4 weeks of either SIT (4-7 × 30 s sprints at 200% Wmax, 3 days week) or MICT (40-60 min cycling at ~65% VO2peak, 5 days per week), and muscle biopsies were obtained pre- and post-training. RESULTS: Training increased PLIN2 (SIT 90%, MICT 68%) and PLIN5 (SIT 47%, MICT 34%) expression in type I fibres only, and increased PLIN3 expression in both type I (SIT 63%, MICT 67%) and type II fibres (SIT 70%, MICT 160%) (all P<0.05). Training did not change LD content but increased the proportion of LD in contact with mitochondria (SIT 12%, MICT 21%, P<0.01). Ceramides were reduced following training (SIT -10%, MICT -7%, P<0.05), but DAG was unchanged. No training × group interactions were observed for any variables. CONCLUSIONS: These results confirm the hypothesis that SIT and MICT results in remodelling of LDs and lowers ceramide concentrations in skeletal muscle of sedentary obese males.

Out-of-pocket expenditure in accessing healthcare services among Chronic Kidney Disease patients in Anuradhapura District

Introduction: Chronic Kidney Disease (CKD) has significant economic impact on both patients' households and the country. Objectives: To assess the out-of-pocket (OOP) expenditure of accessing health services among CKD patients in Anuradhapura District Methods: This community based cross-sectional study included a representative sample of 1174 registered CKD patients from all 19 Medical Officer of Health areas in the District of Anuradhapura. Trained para-medical staff visited the households and administered an interviewer administered questionnaire to gather information. Results: A total of 1118 CKD patients participated. Mean age was 58.3 (SD 10.8) years. Fifty nine (5.3%) patients had been hospitalized during the six months preceding data collection. The total OOP for a hospital admission for one patient was Rs. 3625 (IQR 1650-8760). Thirty eight (3.4%) patients were on dialysis. The median direct cost per patient for an episode of dialysis was Rs.595 (IQR 415-995) while the median direct cost for a dialysis patient per month was Rs.5490 (IQR 3950-10934). In the study population a total of 1095 (98.0%) had attended clinic at least once during the six months preceding the study. The OOP expenditure for a single clinic visit for one patient was Rs.434 (IQR 200- 860). Conclusions: CKD patients living in the Anuradhapura District spent significant amounts on accessing health care which can worsen their economic hardships. Planned interventions are warranted in order to improve their quality of life and financial situation.

Resistance training increases skeletal muscle oxidative capacity and net intramuscular triglyceride breakdown in type I and II fibres of sedentary males.

Recent in vitro and in vivo experimental observations suggest that improvements in insulin sensitivity following endurance training are mechanistically linked to increases in muscle oxidative capacity, intramuscular triglyceride (IMTG) utilization during endurance exercise and increases in the content of the lipid droplet-associated perilipin 2 (PLIN2) and perilipin 5 (PLIN5). This study investigated the hypothesis that similar adaptations may also underlie the resistance training (RT)-induced improvements in insulin sensitivity. Thirteen sedentary men (20 ± 1 years old; body mass index 24.8 ± 0.8 kg m(-2)) performed 6 weeks of whole-body RT (three times per week), and changes in peak O2 uptake (in millilitres per minute per kilogram) and insulin sensitivity were assessed. Muscle biopsies (n = 8) were obtained before and after 60 min steady-state cycling at ~65% peak O2 uptake. Immunofluorescence microscopy was used to assess changes in oxidative capacity (measured as cytochrome c oxidase protein content), IMTG and PLIN2 and PLIN5 protein content. Resistance training increased peak O2 uptake (by 8 ± 3%), COX protein content (by 46 ± 13 and 61 ± 13% in type I and II fibres, respectively) and the Matsuda insulin sensitivity index (by 47 ± 6%; all P < 0.05). In type I fibres, IMTG (by 52 ± 11%; P < 0.05) and PLIN2 content (by 107 ± 19%; P < 0.05) were increased and PLIN5 content tended to increase (by 54 ± 22%; P = 0.054) post-training. In type II fibres, PLIN2 content increased (by 57 ± 20%; P < 0.05) and IMTG (by 46 ± 17%; P = 0.1) and PLIN5 content (by 44 ± 24%; P = 0.054) tended to increase post-training. Breakdown of IMTG during moderate-intensity exercise was greater in both type I and type II fibres (by 43 ± 5 and 37 ± 5%, respectively; P < 0.05) post-RT. The results confirm the hypothesis that RT enhances muscle oxidative capacity and increases IMTG breakdown and the content of PLIN2 and PLIN5 in both type I and type II fibres during endurance-type exercise.

Emerging Epigenetic Targets and Their Molecular Impact on Vascular Remodeling in Pulmonary Hypertension.

Pulmonary Hypertension (PH) is a terminal disease characterized by severe pulmonary vascular remodeling. Unfortunately, targeted therapy to prevent disease progression is limited. Here, the vascular cell populations that contribute to the molecular and morphological changes of PH in conjunction with current animal models for studying vascular remodeling in PH will be examined. The status quo of epigenetic targeting for treating vascular remodeling in different PH subtypes will be dissected, while parallel epigenetic threads between pulmonary hypertension and pathogenic cancer provide insight into future therapeutic PH opportunities.

Sprint interval and traditional endurance training increase net intramuscular triglyceride breakdown and expression of perilipin 2 and 5.

Intramuscular triglyceride (IMTG) utilization is enhanced by endurance training (ET) and is linked to improved insulin sensitivity. This study first investigated the hypothesis that ET-induced increases in net IMTG breakdown and insulin sensitivity are related to increased expression of perilipin 2 (PLIN2) and perilipin 5 (PLIN5). Second, we hypothesized that sprint interval training (SIT) also promotes increases in IMTG utilization and insulin sensitivity. Sixteen sedentary males performed 6 weeks of either SIT (4-6, 30 s Wingate tests per session, 3 days week(-1)) or ET (40-60 min moderate-intensity cycling, 5 days week(-1)). Training increased resting IMTG content (SIT 1.7-fold, ET 2.4-fold; P < 0.05), concomitant with parallel increases in PLIN2 (SIT 2.3-fold, ET 2.8-fold; P < 0.01) and PLIN5 expression (SIT 2.2-fold, ET 3.1-fold; P < 0.01). Pre-training, 60 min cycling at ∼65% pre-training decreased IMTG content in type I fibres (SIT 17 ± 10%, ET 15 ± 12%; P < 0.05). Following training, a significantly greater breakdown of IMTG in type I fibres occurred during exercise (SIT 27 ± 13%, ET 43 ± 6%; P < 0.05), with preferential breakdown of PLIN2- and particularly PLIN5-associated lipid droplets. Training increased the Matsuda insulin sensitivity index (SIT 56 ± 15%, ET 29 ± 12%; main effect P < 0.05). No training × group interactions were observed for any variables. In conclusion, SIT and ET both increase net IMTG breakdown during exercise and increase in PLIN2 and PLIN5 protein expression. The data are consistent with the hypothesis that increases in PLIN2 and PLIN5 are related to the mechanisms that promote increased IMTG utilization during exercise and improve insulin sensitivity following 6 weeks of SIT and ET.

Preferential utilization of perilipin 2-associated intramuscular triglycerides during 1 h of moderate-intensity endurance-type exercise.

The lipid droplet (LD)-associated protein perilipin 2 (PLIN2) appears to colocalize with LDs in human skeletal muscle fibres, although the function of PLIN2 in the regulation of intramuscular triglyceride (IMTG) metabolism is currently unknown. Here we investigated the hypothesis that the presence of PLIN2 in skeletal muscle LDs is related to IMTG utilisation during exercise. We therefore measured exercise-induced changes in IMTG and PLIN2 distribution and changes in their colocalization. Muscle biopsies from the vastus lateralis were obtained from seven lean, untrained men (22 ± 2 years old, body mass index 24.2 ± 0.9 kg m(-2) and peak oxygen uptake 3.35 ± 0.13 l min(-1)) before and after 1 h of moderate-intensity cycling at ~65% peak oxygen uptake. Cryosections were stained for perilipin 2, IMTG and myosin heavy chain type I and viewed using wide-field and confocal fluorescence microscopy. Exercise induced a 50 ± 7% decrease in IMTG content in type I fibres only (P < 0.05), but no change in PLIN2 content. Colocalization analysis showed that the fraction of PLIN2 associated with IMTG was 0.67 ± 0.03 before exercise, which was reduced to 0.51 ± 0.01 postexercise (P < 0.05). Further analysis revealed that the number of PLIN2-associated LDs was reduced by 31 ± 10% after exercise (P < 0.05), whereas the number of PLIN2-null LDs was unchanged. No such changes were seen in type II fibres. In conclusion, this study shows that PLIN2 content in skeletal muscle is unchanged in response to a single bout of endurance exercise. Furthermore, the PLIN2 and IMTG association is reduced postexercise, apparently due to preferential utilization of PLIN2-associated LDs. These results confirm the hypothesis that the PLIN2 association with IMTG is related to the utilization of IMTG as a fuel during exercise.

Integrating epigenetics and metabolomics to advance treatments for pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating vascular disease with multiple etiologies. Emerging evidence supports a fundamental role for epigenetic machinery and metabolism in the initiation and progression of PAH. Here, we summarize emerging epigenetic mechanisms that have been identified as contributors to PAH evolution, specifically, DNA methylation, histone modifications, and microRNAs. Furthermore, the interplay between epigenetics with metabolism is explored while new crosstalk targets to be investigated in PAH are proposed that highlight multi-omics strategies including integrated epigenomics and metabolomics. Therapeutic opportunities and challenges associated with epigenetics and metabolomics in PAH are examined, highlighting the role that epigenetics and metabolomics have in facilitating early detection, personalized dietary plans, and advanced drug therapy for PAH.

Mechanistic regulation of SPHK1 expression and translocation by EMAP II in pulmonary smooth muscle cells.

Phosphorylation of sphingosine by sphingosine kinase 1 (SPHK1) produces the bioactive sphingolipid sphingosine-1-phosphate (S1P), a microvascular and immuno-modulator associated with vascular remodeling in pulmonary arterial hypertension (PAH). The low intracellular concentration of S1P is under tight spatial-temporal control. Molecular mechanisms that mediate S1P burden and S1P regulation of vascular remodeling are poorly understood. Similarities between two early response pro-inflammatory cytokine gene transcript activation profiles, S1P and Endothelial Monocyte Activating Polypeptide II (EMAP II), suggested a strategic link between their signaling pathways. We determined that EMAP II triggers a bimodal phosphorylation, transcriptional regulation and membrane translocation of SPHK1 through a common upstream process in both macrophages and pulmonary artery smooth muscle cells (PASMCs). EMAP II initiates a dual function of ERK1/2: phosphorylation of SPHK1 and regulation of the transcription factor EGR1 that induces expression of SPHK1. Activated ERK1/2 induces a bimodal phosphorylation of SPHK1 which reciprocally increases S1P levels. This identified common upstream signaling mechanism between a protein and a bioactive lipid initiates cell specific downstream signaling representing a multifactorial mechanism that contributes to inflammation and PASMC proliferation which are cardinal histopathological phenotypes of PAH.

How do novel and conventional agri-food wastes, co-products and by-products improve soil functions and soil quality?

Agriculture is estimated to generate about 700 million tons of waste annually in the EU. Novel valorization technologies are developing continuously to recover and recycle valuable compounds and nutrients from waste materials. To close the nutrient loop, low-value agri-food wastes, co-products and by-products (AFWCBs) produced during the valorization process, need to be returned to the soil. However, knowledge on their reaction in soils that is needed to allow efficient and environmentally sound recycling is largely lacking. To this end, we set up a series of laboratory incubation experiments using 10 AFWCBs including insect frass residues made from three different feedstocks, anaerobic digestates from two feedstocks, potato-pulp, rice bran compost, duckweed and two reference crop residues (wheat straw and sugar beet) and measured net N release, C mineralization, dehydrogenase activity (DHA), microbial biomass C (MBC) and community structure. The suppressing potential of frasses and digestates against Rhizoctonia solani was determined using bean. The digestates released the highest net mineral N (50-70%) followed by rice bran compost (55%) and duckweed (30%), while frass made from general food waste and potato-pulp immobilized N like the reference straw for 91 days after incubation. All AFWCBs except digestates significantly increased MBC compared to the control while frasses, potato-pulp and duckweed increased DHA. Frasses and digestates significantly suppressed the development of Rhizoctonia solani in bean plants. AFWCBs from emerging valorizing technologies have the potential to improve microbial activities, C sequestration and may play a significant role in closing the nutrient loop.

Epicardial adipose tissue as a source of nuclear factor-kappaB and c-Jun N-terminal kinase mediated inflammation in patients with coronary artery disease.

CONTEXT: Visceral adipose tissue (AT) is known to confer a significantly higher risk of type 2 diabetes and cardiovascular disease. Epicardial AT has been shown to be related to cardiovascular disease and myocardial function through unidentified mechanisms. Epicardial AT expresses an inflammatory profile of proteins; however, the mechanisms responsible are yet to be elucidated. OBJECTIVES: The objectives of the study were to: 1) examine key mediators of the nuclear factor-kappaB (NFkappaB) and c-Jun N-terminal kinase (JNK) pathways in paired epicardial and gluteofemoral (thigh) AT from coronary artery disease (CAD) and control patients and 2) investigate circulating endotoxin levels in CAD and control subjects. DESIGN: Serums and AT biopsies (epicardial and thigh) were obtained from CAD (n = 16) and non-CAD (n = 18) patients. Inflammation was assessed in tissue and serum samples through Western blot, real-time PCR, ELISAs, and activity studies. RESULTS: Western blotting showed epicardial AT had significantly higher NFkappaB, inhibitory-kappaB kinase (IKK)-gamma, IKKbeta, and JNK-1 and -2 compared with thigh AT. Epicardial mRNA data showed strong correlations between CD-68 and toll-like receptor-2, toll-like receptor-4, and TNF-alpha. Circulating endotoxin was elevated in patients with CAD compared with matched controls [CAD: 6.80 +/- 0.28 endotoxin unit(EU)/ml vs. controls: 5.52 +/- 0.57 EU/ml; P<0.05]. CONCLUSION: Epicardial AT from patients with CAD shows increased NFkappaB, IKKbeta, and JNK expression compared with both CAD thigh AT and non-CAD epicardial AT, suggesting a depot-specific as well as a disease-linked response to inflammation. These studies implicate both NFkappaB and JNK pathways in the inflammatory profile of epicardial AT and highlight the role of the macrophage in the inflammation within this tissue.

Assessing ecological integrity in marine waters, using multiple indices and ecosystem components: challenges for the future.

During the last decade, there have been substantial scientific advances in the development of indices that measure the condition of biological ecosystem elements in coastal and estuarine waters. Though successful, these advances were only the initial steps and a special session on use of indices in ecological integrity assessments was held at the Coastal and Estuarine Research Federation meeting to focus the field on the most appropriate directions for the next decade. The session identified four primary scientific challenges: (i) reduce the array of indices by identifying the index approaches that are most widely successful; (ii) establish minimum criteria for index validation; (iii) intercalibrate methods to achieve uniform assessment scales across geographies and habitats; and (iv) integrate indices across ecosystem elements. Where an explosion of indices characterized the last decade, the next decade needs to be characterized by consolidation. With increased knowledge and understanding about the strengths and weaknesses of competing index approaches, the field needs to unify approaches that provide managers with the simple answers they need to use ecological condition information effectively and efficiently.

Radial forearm free flap as the last resort for oesophageal reconstruction.

Strategies for oesophageal reconstruction following resection vary according to the nature of the pathology encountered, patient factors and surgeon preference. However, reconstruction in patients with multiple previous failed attempts poses specific management challenges. We present the case of a 61-year-old man who underwent oesophageal reconstruction with a radial forearm flap as a last resort.

Values of molecular markers in the differential diagnosis of thyroid abnormalities.

INTRODUCTION: Thyroid cancer (TC), follicular adenoma (FA) and Hashimoto's thyroiditis (HT) are three of the most frequently reported abnormalities that affect the thyroid gland. A frequent co-occurrence along with similar histopathological features is observed between TC and FA as well as between TC and HT. The conventional diagnostic methods such as histochemical analysis present complications in differential diagnosis when these abnormalities occur simultaneously. Hence, the authors recognize novel methods based on screening genetic defects of thyroid abnormalities as viable diagnostic and prognostic methods that could complement the conventional methods. METHODS: We have extensively reviewed the existing literature on TC, FA and HT and also on three genes, namely braf, nras and ret/ptc, that could be used to differentially diagnose the three abnormalities. Emphasis was also given to the screening methods available to detect the said molecular markers. RESULTS AND CONCLUSION: It can be conferred from the analysis of the available data that the utilization of braf, nras and ret/ptc as markers for the therapeutic evaluation of FA and HT is debatable. However, molecular screening for braf, nras and ret/ptc mutations proves to be a conclusive method that could be employed to differentially diagnose TC from HT and FA in the instance of a suspected co-occurrence. Thyroid cancer patients can be highly benefited from the screening for the said genetic markers, especially the braf gene due to its diagnostic value as well as due to the availability of personalized medicine targeted specifically for braf mutants.

An organ culture model for examining epidermal desquamation.

The use of organ cultures of mouse ear skin for examining mechanisms of stratum corneum shedding has been investigated. The formation and loss of stratum corneum cells was assessed by electron microscopic autoradiography and from counts of stratum corneum cell layers taken from sections of plastic-embedded specimens. Using media supplemented with cortisone, there was a linear accumulation of stratum corneum cell layers and the rate of epidermal cell formation corresponded approximately to that in vivo. Cells that accumulated at the stratum corneum surface were loose and could consistently be removed by treatment with detergent and ultrasonication indicating the persistence of shedding mechanisms in vitro.

Lipid composition of cohesive and desquamated corneocytes from mouse ear skin.

An organ culture system has been used to examine differences in the lipid compositions of materials derived from cohesive and desquamated mouse ear stratum corneum. Within this culture system, skin explants display rates of cell replication and differentiation comparable to those observed in vivo for up to 2 weeks and, during this period, loosened or dishesive material accumulates at the surface. Lipid compositions were determined for both intact and loosened stratum corneum derived from cultured skin and also for freshly prepared stratum corneum. In all 3 cases, the profiles of the nonpolar lipids and the ceramides were essentially the same; some of the nonpolar lipids appeared to be of sebaceous origin. The only changes detected upon desquamation were reductions of cholesteryl sulfate and a second unidentified lipid of similar polarity. Cholesteryl sulfate constitutes 4-5% of the polar lipid in fresh stratum corneum or stratum corneum from organ culture. This is reduced to 0.4% in the desquamated material which accumulates in the culture system. The unidentified lipid decreases from 1-2% of the polar lipid in intact fresh or cultured stratum corneum to 0.1% in the desquamated material. The possible function of cholesteryl sulfate in corneocyte cohesion is discussed.

A new face on apoptosis: death-associated protein 3 and PDCD9 are mitochondrial ribosomal proteins.

Two proteins known to be involved in promoting apoptosis in mammalian cells have been identified as components of the mammalian mitochondrial ribosome. Proteolytic digestion of whole mitochondrial ribosomal subunits followed by analysis of the peptides present using liquid chromatography-tandem mass spectrometry revealed that the proapoptotic proteins, death-associated protein 3 (DAP3) and the programmed cell death protein 9, are both components of the mitochondrial ribosome. DAP3 has motifs characteristic of guanine nucleotide binding proteins and is probably the protein that accounts for the nucleotide binding activity of mammalian mitochondrial ribosomes. The observations reported here implicate mitochondrial protein synthesis as a major component in cellular apoptotic signaling pathways.

Some oral carcinomas from Sri Lankan betel/tobacco chewers overexpress p53 oncoprotein but lack mutations in exons 5-9.

We reported a low prevalence (11%) of p53 expression detected by immunohistochemistry in oral squamous cell carcinomas associated with betel/tobacco chewing Sri Lankans (23). Five neoplasms which over-expressed p53 protein were used in the present study of mutations. Despite extensive sequence analysis no mutations were detected in exons 5 through 9 of the p53 gene in all the DNA samples of these neoplasms. Thus, over-expression of p53 is not necessarily synonymous with mutations in the exons studied, although mutations of this gene have been reported in oral and head and neck cancer from Japan and USA. The absence of mutations in betel/tobacco related carcinomas in this population may derive from differences in aetiology, carcinogen metabolism and susceptibility, DNA repair mechanisms and/or genetic predisposition.

Using DNA and hemoglobin adducts to improve the risk assessment of butadiene.

The purpose of this paper is to review what we know about various biomarkers of butadiene in animal, human and in vitro studies, and to draw inferences from these data that impact on the accurate assessment of human risks for cancer. Studies comparing the DNA and hemoglobin adducts of butadiene with exposure, metabolism and genotoxicity have provided a great deal of insight that is applicable to biologically based risk assessment. First, the DNA and hemoglobin adduct data strongly support the conclusion that 3,4-epoxy-1,2-butanediol is the major electrophile available for binding to these macromolecules. Biomarker studies have also provided insight into the possibility of a sensitive population associated with the GSTT1 null genotype. While it is clear that lymphocytes from GSTT1 null individuals are more sensitive for the induction of sister chromatid exchanges (SCE) following in vitro exposure to 1,2,3,4-diepoxybutane, there was no such increase in SCE or other biomarkers of genotoxicity in workers exposed to 1-3 p.p.m. butadiene, regardless of GST genotype. The globin adduct data also demonstrate that there is roughly a tenfold range for interindividual differences in the metabolism of butadiene. This type of analysis represents an excellent means for providing scientific data for this critical determinant. Another useful application of hemoglobin adducts in risk assessment was demonstrated by regressing data for various endpoints for genotoxicity against that individual's biologically effective dose, thereby providing an independent mechanism for evaluation that excludes any possible confounding by inappropriate controls. Finally, biomarker studies have identified critical gaps in our knowledge that are needed for the accurate assessment of butadiene. Most notable of these is the lack of diepoxide-specific biomarkers in mice, rats and humans.