Interactions between Crassostrea virginica larvae and Deepwater Horizon oil: Toxic effects via dietary exposure.

The Deepwater Horizon (DWH) disaster released crude oil in the Gulf of Mexico for 87 days, overlapping with the reproductive season and recruitment of the oyster Crassostrea virginica. The pelagic larval life stages of C. virginica are particularly vulnerable to contaminants such as polycyclic aromatic hydrocarbons (PAHs) and oil droplets. Based on their lipophilic properties, PAHs and oil droplets can adsorb onto phytoplankton and filter-feeding C. virginica larvae may be exposed to these contaminants bound to suspended sediment, adsorbed onto algal and other particles, or in solution. This study examined the effects of exposure of C. virginica larvae to algae mixed with DWH oil. In a 14-day laboratory exposure, 5 day-old C. virginica larvae were exposed to Tisochrysis lutea mixed with four concentrations of unfiltered DWH oil (HEWAF) in a static renewal system. Larval growth, feeding capacity, abnormality and mortality were monitored throughout the exposure. Total PAH (n = 50) content of the water medium, in which larvae were grown, were quantified by GC/MS-SIM. Oil droplets were observed bound to algae, resulting in particles in the size-range of food ingested by oyster larvae (1-30 µm). After 14 days of exposure, larval growth and survival were negatively affected at concentrations of tPAH50 as low as 1.6 µg L-1. GC/MS-SIM analysis of the exposure medium confirmed that certain PAHs were also adsorbed by T. lutea and taken up by oyster larvae via ingestion of oil droplets and/or contaminated algae. Long-term exposure to chronic levels of PAH (1.6-78 µg tPAH50 L-1) was shown to negatively affect larval survival. This study demonstrates that dietary exposure of oyster larvae to DWH oil is a realistic route of crude oil toxicity and may have serious implications on the planktonic community and the food chain.

Sensitivity of eastern oyster (Crassostrea virginica) spermatozoa and oocytes to dispersed oil: Cellular responses and impacts on fertilization and embryogenesis.

The 2010 Deepwater Horizon (DWH) oil spill released millions of barrels of oil and dispersant into the Gulf of Mexico. The timing of the spill coincided with the spawning season of Crassostrea virginica. Consequently, gametes released in the water were likely exposed to oil and dispersant. This study aimed to (i) evaluate the cellular effects of acute exposure of spermatozoa and oocytes to surface slick oil, dispersed mechanically (HEWAF) and chemically (CEWAF), using flow-cytometric (FCM) analyses, and (ii) determine whether the observed cellular effects relate to impairments of fertilization and embryogenesis of gametes exposed to the same concentrations of CEWAF and HEWAF. Following a 30-min exposure, the number of spermatozoa and their viability were reduced due to a physical action of oil droplets (HEWAF) and a toxic action of CEWAF respectively. Additionally, reactive oxygen species (ROS) production in exposed oocytes tended to increase with increasing oil concentrations suggesting that exposure to dispersed oil resulted in an oxidative stress. The decrease in fertilization success (1-h), larval survival (24-h) and increase in abnormalities (6-h and 24-h) may be partly related to altered cellular characteristics. FCM assays are a good predictor of sublethal effects especially on fertilization success. These data suggest that oil/dispersant are cytotoxic to gametes, which may affect negatively the reproduction success and early development of oysters.

Lethal and sub-lethal effects of Deepwater Horizon slick oil and dispersant on oyster (Crassostrea virginica) larvae.

In April 2010, crude oil was spilled from the Deepwater Horizon (DWH) oil platform for 87 days, coincident with the spawning season and recruitment of the oyster, Crassostrea virginica, in the Gulf of Mexico. Impacts of acute exposures to surface-collected DWH oil (HEWAF), dispersed oil (CEWAF) and dispersant alone (Corexit 9500A(®)) on planktonic larval stages of C. virginica (veliger, umbo and pediveliger) were tested in the laboratory. Exposures to HEWAF, CEWAF and dispersant were toxic to larvae impairing growth, settlement success and ultimately survival. Larval growth and settlement were reduced at concentrations of tPAH50 ranging from 1.7 to 106 µg L(-1) for HEWAF and 1.1-35 µg L(-1) for CEWAF, concentrations well within the range of water sampled during the DWH oil spill. Sublethal effects induced by oil and dispersant could have significant ecological implications on oyster populations and on the whole estuarine ecosystem.

Impacts of Deepwater Horizon oil and associated dispersant on early development of the Eastern oyster Crassostrea virginica.

The explosion of the Deepwater Horizon (DWH) oil platform resulted in large amounts of crude oil and dispersant Corexit 9500A® released into the Gulf of Mexico and coincided with the spawning season of the oyster, Crassostrea virginica. The effects of exposing gametes and embryos of C. virginica to dispersant alone (Corexit), mechanically (HEWAF) and chemically dispersed (CEWAF) DWH oil were evaluated. Fertilization success and the morphological development, growth, and survival of larvae were assessed. Gamete exposure reduced fertilization (HEWAF: EC201h=1650µg tPAH50L(-1); CEWAF: EC201h=19.4µg tPAH50L(-1); Corexit: EC201h=6.9mgL(-1)). CEWAF and Corexit showed a similar toxicity on early life stages at equivalent nominal concentrations. Oysters exposed from gametes to CEWAF and Corexit experienced more deleterious effects than oysters exposed from embryos. Results suggest the presence of oil and dispersant during oyster spawning season may interfere with larval development and subsequent recruitment.

Essential oil from leaves of Cinnamomum osmophloeum acts as a xanthine oxidase inhibitor and reduces the serum uric acid levels in oxonate-induced mice.

The xanthine oxidase (XOD) inhibitory activity and anti-hyperuricemia effect in mice of Cinnamomum osmophloeum, which is an endemic tree in Taiwan, were evaluated in this study. The results demonstrated that the essential oil of C. osmophloeum leaves presented the strongest XOD inhibition activity (IC(50)=16.3 µg/ml); however, no significant XOD inhibition activities were found in ethanolic and hot water extracts. Furthermore, among the main compounds of essential oil, the cinnamaldehyde exhibited the potent XOD inhibition activity with an IC(50)=8.4 µg/ml. Besides, the reducing serum uric acid levels in oxonate-induced mice by cinnamaldehyde were further investigated. The hyperuricemic mice were oral administrated cinnamaldehyde at a dosage of 150 mg/kg, the uric acid value in serum was reduced from 5.25±0.63 to 2.10±0.04 mg/dl, the levels of serum uric acid in mice was lowered down by 84.48% as compared to the hyperuricemic control group. Based on the results obtained in this study, cinnamaldehyde may be a potential lead compound for developing the pharmaceutic for anti-hyperuricemia agent.

Cytoreductive surgery and perioperative intraperitoneal chemotherapy for peritoneal carcinomatosis from colorectal carcinoma: non-mucinous tumour associated with an improved survival.

AIMS: Cytoreductive surgery combined with perioperative intraperitoneal chemotherapy has been reported as a treatment option for patients with peritoneal carcinomatosis from colorectal carcinoma. METHODS: Thirty patients with colorectal peritoneal carcinomatosis underwent cytoreductive surgery and perioperative intraperitoneal chemotherapy. All appendiceal cancers were excluded. All patients were followed until January 2006 or death. Univariate analysis was performed to evaluate significant prognostic factors for overall survival, defined from the time of surgery. RESULTS: There were 13 male patients. The mean age at the time of surgery was 54years. There was no hospital mortality. The mean duration of hospital stay was 27days. The overall median survival was 29months, with 1- and 2-year survival of 72% and 64%, respectively. Twenty-one patients had complete cytoreduction and their 1- and 2-year survival rates were 85% and 71%, respectively. Univariate analysis demonstrated that patients with non-mucinous colorectal adenocarcinoma, Peritoneal Cancer Index (PCI) < or =13, and complete cytoreduction were associated with an improved survival. CONCLUSIONS: This study reported on 30 patients who underwent cytoreductive surgery and perioperative intraperitoneal chemotherapy for colorectal peritoneal carcinomatosis. Patients with mucinous tumour had relatively more extensive intraperitoneal disease. Non-mucinous colorectal adenocarcinoma, PCI < or =13, and complete cytoreduction were associated with an improved survival.

[Study on using colloidal gold immuno-dot assay to detect special antibody of hemorrhagic fever renal syndrome].

OBJECTIVE: To seek for a simple, sensitive and rapid assay to detect specific antibody of patients suffering from hemorrhagic fever renal syndrome (HFRS). METHODS: Serum anti-HFRS IgM (sIgM) and IgG (sIgG) antibody were detected in 186 patients with HFRS using colloidal gold immuno-dot assay (CGIDA), and compared with those detected with enzyme linked immunosorbant assay (ELISA) and indirect fluorescent antibody test (IFAT). Kuhuang Shenmai injection (KHSM) combined Astragalus decoction (AD) treatment was applied to 101 HFRS patients in a treated group (n = 50), and the effect of therapy was compared with that in a control group (n = 51) treated with ribarvirin and ganlixin injection. RESULTS: CGIDA showed positive sIgM antibody in 132 cases (70.9%) and positive sIgG antibody in 163 (87.1%) of the 186 HFRS patients. As compared the two groups in crossing stage, the rate of crossing shock stage in the treated group was higher than that in the control (P < 0.05). As for rate of crossing oliguria stage and from febrile stage directly developed into diuretic stage, the difference between the two groups was insignificant (P < 0.05). CONCLUSION: CGIDA shows the merits of quick, simple and convenient in detecting sIgM and sIgG antibody in HFRS patients. The effect of KHSM combined AD treatment was not significantly different from that of ribarvirin and ganlixin injection.

Effects of surface treatments on shear bond strengths between a resin cement and an alumina core.

STATEMENT OF PROBLEM: Although bonding to all-ceramic restorations is desirable, there is little information on the use of resin cements containing a phosphate monomer, and the importance of different surface treatments on their adhesion to high-strength core materials. PURPOSE: This study attempted to determine the shear bond strength values between Panavia 21 resin cement (Kuraray) and an alumina core material (In-Ceram) after 3 surface treatments and the application of a silane coupling agent. MATERIAL AND METHODS: Forty-five In-Ceram cylindrical rods were fabricated and assigned to 3 groups. Group I specimens were treated with a 9.5% hydrofluoric (HF) acid, group II with a 5% HF acid, and group III were sandblasted. All specimens were coated with a silane coupling agent (Cavex Clearfil Photobond and Activator) before cementation with Panavia 21 to sandblasted nickel-chromium rods. As a control, group IV consisted of 8 porcelain (Vitadur Alpha) rods treated with a 5% HF acid and silane. All specimens were subjected to a load of 1.2 kg during cementation, then stored under water for 36 hours. A jig mounted on a Hounsfield Universal Testing machine was used at a crosshead speed of 0.5 mm/min to test the shear bond strengths. RESULTS: The results were 14.65 +/- 4.64 MPa for group I, 18.03 +/- 6.13 MPa for group II, and 22.35 +/- 5.98 MPa for group III In-Ceram specimens; and 18.05 +/- 8.46 MPa for control (group IV). CONCLUSION: The use of Panavia 21 resin cement and a silane coupling agent can achieve a successful bond between either sandblasted or 5% HF acid-etched In-Ceram core material.

Assessing potential health risks from microcystin toxins in blue-green algae dietary supplements.

The presence of blue-green algae (BGA) toxins in surface waters used for drinking water sources and recreation is receiving increasing attention around the world as a public health concern. However, potential risks from exposure to these toxins in contaminated health food products that contain BGA have been largely ignored. BGA products are commonly consumed in the United States, Canada, and Europe for their putative beneficial effects, including increased energy and elevated mood. Many of these products contain Aphanizomenon flos-aquae, a BGA that is harvested from Upper Klamath Lake (UKL) in southern Oregon, where the growth of a toxic BGA, Microcystis aeruginosa, is a regular occurrence. M. aeruginosa produces compounds called microcystins, which are potent hepatotoxins and probable tumor promoters. Because M. aeruginosa coexists with A. flos-aquae, it can be collected inadvertently during the harvesting process, resulting in microcystin contamination of BGA products. In fall 1996, the Oregon Health Division learned that UKL was experiencing an extensive M. aeruginosa bloom, and an advisory was issued recommending against water contact. The advisory prompted calls from consumers of BGA products, who expressed concern about possible contamination of these products with microcystins. In response, the Oregon Health Division and the Oregon Department of Agriculture established a regulatory limit of 1 microg/g for microcystins in BGA-containing products and tested BGA products for the presence of microcystins. Microcystins were detected in 85 of 87 samples tested, with 63 samples (72%) containing concentrations > 1 microg/g. HPLC and ELISA tentatively identified microcystin-LR, the most toxic microcystin variant, as the predominant congener.

Surface roughness and flexural strength of self-glazed, polished, and reglazed In-Ceram/Vitadur Alpha porcelain laminates.

PURPOSE: Because of existing controversy, the present study investigated 3 methods for reducing surface roughness and improving the strength of porcelain restorations. MATERIALS AND METHODS: Ninety laminated In-Ceram/Vitadur Alpha self-glazed porcelain disks were fabricated and randomly divided into 3 groups (n = 30 each). Group 1 consisted of 30 of the original disks. The remaining 60 disks were then polished by 6 operators according to the recommendations of the American Academy of Esthetic Dentistry; group 2 consisted of 30 of these polished disks. The remaining 30 polished disks were reglazed (group 3). Average roughness values (Ra) of the veneers were measured by a profilometer. In each group, 20 disks were subjected to a flexure test, with 10 of the In-Ceram cores and 10 of the Vitadur Alpha veneers placed in tension. RESULTS: The Ra values were 0.5 +/- 0.1 micron, 0.7 +/- 0.3 micron, and 0.4 +/- 0.1 micron for groups 1 to 3, respectively. Following one-way analysis of variance (ANOVA), Bonferroni's multiple comparison tests found that groups 1 and 3 were significantly smoother than the polished group (P < 0.001). Reglazed disks were also significantly smoother than the original self-glazed disks (P < 0.01). With the veneers in tension, the flexural strengths were 151 +/- 22 MPa, 118 +/- 22 MPa, and 172 +/- 27 MPa for group 1 to 3, respectively. Following one-way ANOVA, Bonferroni's multiple comparison tests found that groups 1 and 3 were similar and were significantly stronger than the polished samples (P < 0.05 and P < 0.001). CONCLUSION: Reglazing polished porcelain surfaces significantly improved the surface texture and flexural strength of the materials tested.

[Effect of inflammatory factor and yishen huoxue xiezhuo decoction on growth of glomerular mesangial cells in rats].

OBJECTIVE: To explore the part of inflammatory factors (cytokines) and Yishen Huoxue Xiezhuo (YSHXXZ) Decoction on the proliferation of extracorporeal cultured mesangial cells (MCs) in rats. METHODS: The effects of lipopolysaccharide (LPS) and interleukin-6 (IL-6) as stimulating factors, its action on the proliferation of rat MCs were investigated by using the technique of 3H-TdR incorporation. Meanwhile, adopting serum pharmacology assay, the medicated serum of rat, containing YSHXXZ decoction was extracted and its effects on the growth of MCs were also studied. RESULTS: LPS and IL-6, in a dose-dependent and time-dependent manner, could markedly stimulate the MCS proliferation, while this stimulatory effects could be strongly antagonized by the serum containing YSHXXZ decoction. CONCLUSION: Mesangial cell is the main target cell of the action of YSHXXZ decoction, and the inhibition on MCs might be one of the mechanisms of the YSHXXZ decoction in preventing the progression of chronic glomerulonephritis.

Selenium-dependent glutathione peroxidase-GI is a major glutathione peroxidase activity in the mucosal epithelium of rodent intestine.

Gpx2 mRNA, encoding a selenium-dependent glutathione peroxidase (GPX-GI), has been found to be highly expressed in the gastrointestinal tract (GI) mucosal epithelium. In this study, we show that GPX-GI is produced in the mucosal epithelium of the adult rat GI tract and that the activity levels are comparable to that from GPX-1. Post-mitochondrial supernatant GPX activity from the mucosal epithelium of the complete length of the small intestine was partially purified. A sample enriched for putative GPX-GI was fractionated by SDS-polyacrylamide gel electrophoresis. Polypeptides of 21 kDa and 22 kDa were digested with trypsin. After resolving the tryptic peptides by high pressure liquid chromatography (HPLC), the major peaks were analyzed for their amino acid sequence by Microflow-HPLC-Tandem Mass Spectrometry and automated Edman degradation sequencing. Both methods revealed that the 21-kDa sample contained rat GPX-GI determined by the sequence homology with the deduced mouse GPX-GI polypeptide sequence. Rat GPX-1 was also detected in the samples. AntiGPX-GI and antiGPX-1 antibodies were used to determine the distribution of the respective isoenzyme activities along the length of the intestine and with respect to the crypt to villus axis in rats. GPX-GI and GPX-1 activities were uniformly distributed in the middle and lower GI tract and with respect to the crypt to villus axis. GPX-GI activity accounted nearly the same percentage of the total GPX activity as GPX-1 in all of the these compartments. Studies on the distal ileum segment of wildtype and Gpx1 gene knockout mice showed that GPX-GI activity was also at parity with GPX-1 in the mucosal epithelium of this segment.

Expression of selenium-dependent glutathione peroxidase in human breast tumor cell lines.

In estrogen receptor (ER)-positive breast cancer cell lines, very low expression of glutathione peroxidase-1 (GPX-1) activity and hgpx1 mRNA has been observed. Such cell lines have been used as models in studies of resistance to redox cycling anticancer drugs. In particular, large increases in GPX-1 activity levels by expression of transfected GPX-1 cDNA have been shown to confer some resistance to such drugs. It has never been determined that such low GPX-1 expression is a common feature of breast cancer. Based on previous limited surveys of breast cancer cell lines, it has been suggested that there may be an inverse correlation between ER status and GPX-1 production. Here we report the results from a larger survey of breast cancer cell lines, including six recently isolated cell lines. A near absence of hgpx1 mRNA expression was observed in 3 of 13 ER-negative cell lines; 1 of 4 ER-positive cell lines had high production of GPX-1. Both observations weaken the proposed inverse correlation between ER status and GPX-1 production. We have evidence to suggest that one cell line, COH-BR-5 (ER-negative), lacked hgpx1 gene expression prior to culture. This is based on the finding of stable hgpx1 gene expression during serial culture of ER-negative breast cancer cell lines newly isolated from malignant effusion and absence of hgpx1 mRNA expression in COH-BR-5. Expression of hgpx2 mRNA (producing GPXGI, the GI tract GPX) was detected in several long and newly established, ER-negative breast cancer cell lines. Cell lines, COH-BR-5 and MDA-MB-175, expressed only hgpx2 mRNA. The hgpx2 mRNA was detected in COH-BR-5 and COH-BR-7 at low passage number, suggesting that hgpx2 gene expression occurs in breast cancer malignant effusion. Thus, studies of the role of GPX in redox drug resistance may account for changes in hgpx2 gene expression. Phospholipid hydroperoxide GPX activity was not found to be generally elevated above normal tissue levels in newly established breast cancer-derived cell lines.

Cloning and sequencing of the cDNA encoding a human testis phospholipid hydroperoxide glutathione peroxidase.

A human cDNA that encodes a polypeptide that has 94% deduced amino-acid sequence identity to porcine phospholipid hydroperoxide glutathione peroxidase was cloned from a testis library. The sequence shows preservation of the UGA selenocysteine codon, putative active-site Trp and Glu residues and a Tyr residue that is phosphorylated in the porcine protein. The 3'-UTR shows some conservation of sequences implicated in the insertion of selenocysteine at an opal codon in human glutathione peroxidase-1.

Painful right iliac fossa mass caused by a migrating left ovary.

A young woman presenting with right iliac fossa pain was found to have a palpable mass. Ultrasound and computed tomography demonstrated a calcified solid mass, which was extraintestinal on barium enema. Laparotomy confirmed an infarcted left ovarian cyst due to torsion of an attenuated but intact fallopian tube. To our knowledge, this is the first documented case of ovarian autoamputation in evolution. A migrating left ovary should be added to the differential diagnosis of a painful right iliac fossa mass.

Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis.

Aflatoxins are polyketide-derived secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Among the catalytic steps in the aflatoxin biosynthetic pathway, the conversion of sterigmatocystin to O-methylsterigmatocystin and the conversion of dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin are catalyzed by an S-adenosylmethionine-dependent O-methyltransferase. A cDNA library was constructed by using RNA isolated from a 24-h-old culture of wild-type A. parasiticus SRRC 143 and was screened by using polyclonal antiserum raised against a purified 40-kDa O-methyltransferase protein. A clone that harbored a full-length cDNA insert (1,460 bp) containing the 1,254-bp coding region of the gene omt-1 was identified by the antiserum and isolated. The complete cDNA sequence was determined, and the corresponding 418-amino-acid sequence of the native enzyme with a molecular weight of 46,000 was deduced. This 46-kDa native enzyme has a leader sequence of 41 amino acids, and the mature form of the enzyme apparently consists of 377 amino acids and has a molecular weight of 42,000. Direct sequencing of the purified mature enzyme from A. parasiticus SRRC 163 showed that 19 of 22 amino acid residues were identical to the amino acid residues in an internal region of the deduced amino acid sequence of the mature protein. The 1,460-bp omt-1 cDNA was cloned into an Escherichia coli expression system; a Western blot (immunoblot) analysis of crude extracts from this expression system revealed a 51-kDa fusion protein (fused with a 5-kDa beta-galactosidase N-terminal fragment).(ABSTRACT TRUNCATED AT 250 WORDS)

Rectal perforation after barium enema in a patient with cytomegalovirus colitis--a case report.

The very rare complication of rectal perforation during the barium enema of an immunocompetent Chinese man with cytomegalovirus (CMV) colitis is described. Rectal biopsy performed over three weeks earlier was unlikely to have been related to the perforation. Histopathological examination of the colonic mucosa showed features of active chronic colitis and CMV infection. After treatment with Ganciclovir, there was cessation of symptoms with eradication of the virus on repeat biopsy samples. It is suggested that presence of CMV colitis predisposed to rectal perforation. The clinical presentation, course of disease and response to treatment supported the concept of CMV as a primary aetiological agent. This complication should be borne in mind when performing barium enemas in patients with clinical features of colitis, especially if they are immunocompromised.

Phospholipid hydroperoxide glutathione peroxidase is the 18-kDa selenoprotein expressed in human tumor cell lines.

Human tumor cell lines cultured in 75Se-containing media demonstrate four major 75Se-labeled cellular proteins (57, 22, 18, and 12 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Among these selenoproteins, an enzymatic activity is known only for the 22-kDa protein, since this protein has been identified as the monomer of glutathione peroxidase. However, all tested cell lines also contained a peroxidase activity with phospholipid hydroperoxides that is completely accounted for by the other selenoenzyme, phospholipid hydroperoxide glutathione peroxidase (PHGPX) (Ursini, F., Maiorino, M., and Gregolin, C. (1985) Biochim. Biophys. Acta 839, 62-70). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of 75Se-labeled proteins separated by gel permeation chromatography supported the identification of PHGPX as the monomeric protein matching the 18 kDa band. This paper is the first report on the identification of PHGPX in human cells.

Effects of palm carotenoids in rat hepatic cytochrome P450-mediated benzo(a)pyrene metabolism.

Using benzo(a)pyrene (BaP) metabolism as a probe for chemical carcinogenesis, in vitro and in vivo effects of palm-oil carotenoid [beta-carotene (BC), alpha-carotene (AC), or canthaxanthin (CTX)] on BaP metabolism in the rat hepatic cytochrome P450-mediated monooxygenase system were studied. Apparent Michaelis-Menten constants (Km) for formation of the precursor carcinogen, 7,8-dihydrodiol BaP, were found to be 14.4 (BC), 1.74 (AC), and 0.7 (CTX) mumol/L. The order of anticarcinogenic strength established in this study was BC much greater than AC greater than CTX. Increased formation of the detoxification intermediate, 3-hydroxy BaP, with increased carotenoid concentration was observed. The order of detoxification strength was BC greater than AC = CTX. The presence of carotenoids in vivo inhibited BaP metabolism. Using 9,10-dihydrodiol BaP as an indicator for inhibition, the order of the antioxidative activity was palm oil (with carotenoids) greater than BC greater than CTX greater than palm oil (without carotenoids). BC and AC may selectively modify the rat-liver microsomal enzymes, thus providing a biochemical mechanism for the inhibitory effect of palm carotenoids on chemical carcinogenesis.