Four sets of experiments were conducted to examine the influence of conjugated linoleic acid (CLA) isomers during proliferation and differentiation of cultures of 3T3-L1 preadipocytes using physiological culturing conditions. Cultures treated with either albumin [bovine serum albumin (BSA) vehicle] or linoleic acid (LA) served as controls. For the proliferation study (Expt.1), cells were cultured in media containing a crude mixture of CLA isomers or pure LA at 0, 10, 50, or 200 microM for 4 d. Preadipocyte proliferation (cell number, 3H-thymidine incorporation into DNA) decreased as the level of CLA increased in the cultures. In contrast, LA had no impact on DNA synthesis. In Experiment 2a, postconfluent cultures were grown in media containing a crude mixture of CLA isomers or LA at 0, 10, 50, or 200 microM for the next 6 d. Postconfluent cultures supplemented with 50-200 microM CLA had less triglyceride (TG) and were smaller in size than cultures supplemented with similar amounts of LA. In Experiment 2b, postconfluent cultures supplemented with 200 microM of a crude mixture of CLA isomers or LA were harvested on days 1, 3, 6, or 9. Differences in TG content of cultures supplemented with 200 microM CLA compared to control and LA-supplemented cultures became apparent after 3 d of culture. Experiments 3a and 3b examined whether the fatty acid vehicle (BSA vs. ethanol) or the vitamin E status (+/-0.2 mM alpha-tocopherol) of the cultures altered CLA's impact on preadipocyte TG content. In Experiment 3a, ethanol-treated cultures had more TG than non-ethanol-treated cultures regardless of the fatty acid treatment. In Experiment 3b, cultures treated with 100 microM of either a crude mixture of CLA or the trans-10,cis-12 CLA isomer without supplemental vitamin E for 6 d had less TG than CLA-treated cultures containing vitamin E. In Experiment 4, postconfluent cultures were grown in media containing 100 microM LA or either a crude mixture of CLA isomers or the trans-10,cis-12 CLA isomer for 24-96 h to assess CLA's influence on the cell cycle and indices of apoptosis. Cultures treated with 100 microM CLA for 24-96 h had more apoptotic cells than BSA- or LA-treated cultures. Furthermore, cultures treated for 48 h with CLA had fewer cells in the S-phase than control cultures. The effects of the trans-10,cis-12 CLA isomer were more pronounced than those of the crude mixture of CLA isomers. These data suggest that CLA may exert its antiobesity effects by inhibiting proliferation, attenuating TG content, and/or inducing apoptosis in (pre)adipocytes.

No abstract

A series of experiments was conducted using 3T3-L1 preadipocytes as the cell model to determine: (i) whether the triglyceride (TG)-lowering effects of a crude mixture of conjugated linoleic acid (CLA) isomers were due to a specific isomer of CLA and the timing of treatment, (ii) if CLA reduced TG content by inhibiting a key regulator of adipogenesis, (iii) if CLA incorporated into either neutral lipid or phospholipid cell fractions, and (iv) whether the effects of CLA treatment were reversible. Trans-10,cis-12 CLA reduced TG content, whereas the cis-9,trans-11 isomer increased TG content compared to vehicle [bovine serum albumin (BSA)] controls. Treatment with 50 microM trans-10,cis-12 CLA during the entire 6 d of differentiation reduced TG content to a greater extent than treatment during either the first 3 d or last 3 d of differentiation. Trans-10,cis-12 CLA treatment of preadipocyte cultures for 48 h increased peroxisome proliferator activated receptor gamma2 (PPARgamma2) protein expression compared to cultures treated with linoleic acid (LA) or the BSA controls. CLA had no effect on adipose P2 (aP2), a fatty acid-binding protein regulated by PPARgamma2. Both the cis-9,trans-11 and the trans-10,cis-12 isomers of CLA were incorporated into neutral lipids and phospholipids. However, cis-9,trans-11 CLA levels were one- to twofold higher than trans-10,cis-12 CLA levels. Moreover, trans-10,cis-12 CLA treatment reduced cis-11 18:1 concentrations in both neutral lipids and phospholipids while increasing cis-9 18:1 and 18:2 concentrations. Palmitoleic acid (16:1) levels were also lower in the neutral lipid fraction of cultures treated with trans-10,cis-12 CLA. Supplementing trans-10,cis-12 CLA-treated cultures (50 microM) with increasing levels of LA resulted in a dose-dependent increase in TG content compared to cultures treated with 50 microM CLA alone. LA supplementation also prevented some of the morphological changes associated with trans-10,cis-12 CLA treatment as seen with scanning electron microscopy. Treatment with 50 microM trans-10,cis-12 CLA for 6 d decreased PPARgamma2 levels, and supplementation of CLA-treated cultures with LA increased PPARgamma2 levels compared with cultures treated with CLA alone. Taken together, these data indicate that in cultures of 3T3-L1 preadipocytes: (i) trans-10,cis-12 CLA is the TG-lowering isomer of CLA, and its effects are dependent on dose, duration of treatment, and the amount of LA in the cultures; (ii) trans-10,cis-12 CLA treatment alters the monounsaturated fatty acid profile of neutral- and phospholipids of the cultures; and (iii) although acute (2-d) trans-10,cis-12 CLA treatment increased PPARgamma2 protein levels, chronic (6-d) treatment decreased PPARgamma2 levels.

Despite the extensive use of aquatic insects to evaluate freshwater ecosystem health, little is known about the underlying factors that result in sensitivity differences between taxa. Organismal characteristics (respiratory strategy and body size) were used to explore the rates of [3H]H2O and [14C]chlorpyrifos accumulation in aquatic insects. Ten aquatic insect taxa, including ephemeropteran, trichopteran, dipteran, hemipteran, and coleopteran species, were exposed to [14C]chlorpyrifos (240 ng·L–1) and [3H]H2O for up to 12 h. Because exchange epithelial surfaces on the integument are permeable to water, [3H]H2O was used as a quantitative surrogate for exposed cellular surface area. [14C]Chlorpyrifos uptake rates were highly correlated with water permeability in all 10 taxa tested and largely covaried with body size and respiratory strategy. Rates were highest among smaller organisms on a per-weight basis and in taxa with relatively large external cellular surfaces such as gills. Air-breathing taxa were significantly less permeable to both [3H]H20 and [14C]chlorpyrifos. A method for labeling exposed epithelial surfaces with a fluorescent dye was developed. This technique allowed discrimination between exchange epithelium and barrier tissue on the integument. Fluorescent dye distributions on the body surface provided a rapid method for estimating exposed epithelium consistent with [3H]H20 and [14C]chlorpyrifos accumulation.

This review focuses on cellular events that modulate hepatotoxicity subsequent to initial liver insult. Cellular events that determine the nature and extent of hepatotoxic injury and the ultimate outcome of that injury are also discussed. The roles of cell types other than hepatocytes, hepatocyte organelle-specific processes, and regeneration in progression or recovery from liver injury are emphasized. Leukocyte activities are key events in two distinct hepatotoxicities. Neutrophil-mediated, periportal inflammation appears to play a primary role in progression of alpha-naphthylisothiocyanate-induced cholangiolitic hepatitis. However, a humorally mediated autoimmune response to protein adducts that occurs after anesthesia is critical in onset of halothane-induced hepatitis. New insights into specific events at the hepatocyte level are also emerging. Although reducing gap junctional communication between hepatocytes can protect against progression of liver injury, down-regulation of the subunit proteins (connexins) can isolate neoplastic cells from growth regulation. Acidic intracellular pH characteristic of hypoxia is protective against both hypoxic and toxicant-induced cell injury. In oxidative injury, a pH-mediated mitochondrial permeability transition causes mitochondrial uncoupling and ATP loss and leads to cell death. The ultimate outcome of hepatotoxic injury depends on the extent of tissue repair. Stimulation of tissue repair after a sublethal dose of CCl4 appears to be the central mechanism in protection against death from a subsequent large dose. Taken together, these examples illustrate the importance of events subsequent to initial liver injury as determinants of extent of liver damage.

A high prevalence of vertebral deformities has been observed in various fishes, especially cyprinids, from certain regions of the Willamette River for many years. One proposed source of these deformities is exposure to toxicants. Histological evaluation of affected chiselmouth Acrocheilus alutaceus revealed that all lesions associated with vertebrae were associated with metacercariae of digenean trematodes. Approximately half of the northern pikeminnow Ptychocheilus oregonensis had infections in which metacercariae were associated with these lesions. Metacercariae were also associated with vertebral lesions in three of four affected peamouth Mylocheilus caurinus. Many metacercariae that were present within the vertebral bodies were associated with bony dysplasia and bony proliferation in all three species. We also evaluated the association of the metacercariae with the vertebral deformities, using intact fish that had been cleared with trypsin. Fish from the affected regions had a much higher prevalence of metacercariae and deformities and a greater abundance of metacercariae than those in the reference site. Chiselmouths had more deformities and metacercariae than northern pikeminnow. In all fish species, 77% of deformities were directly associated with metacercariae; in chiselmouths, about 95% of the deformities exhibited this relationship. Two types of metacercariae were identified in affected fish: Apophallus sp. (Heterophyidae) and a neascus type (Strigeidida). The Apophallus sp. appeared to be more closely associated with the skeleton deformities. A Myxobolus sp. morphologically similar to M. cyprini was also associated with the vertebral lesions in about 50% of the northern pikeminnow and 5% of the chiselmouths. Intact plasmodia were found in somatic muscle, and lesions containing free spores were often located at bone surfaces. This survey demonstrates that metacercariae (probably Apophallus sp.) and a Myxobolus sp. are major causes of the vertebral deformities seen in cyprinid fishes from certain regions of the Willamette River.

Fish in agricultural and remote areas may be exposed to endosulfan and its degradation products as a result of direct runoff, atmospheric transport and deposition. The following study used the zebrafish developmental model to investigate the responses to endosulfan I and endosulfan sulfate, the major degradation product of endosulfan I and II. Embryos were dechorionated and waterborne exposed to the endosulfan I or endosulfan sulfate from 6 to 120 hours post fertilization (hpf). Endosulfan I exposure concentrations ranged from 0.01 to 10 μg/L and endosulfan sulfate from 1 to 100 μg/L. Water solutions were renewed every 24 hours and fish were scored for overt developmental and behavioral abnormalities. Chemical analysis was performed on water, whole embryo, and larvae samples to determine waterborne exposure concentrations and tissue concentrations throughout the 5-day period. The most sensitive toxicity endpoint for both endosulfan I and endosulfan sulfate was an abnormal response of the embryo/larvae to touch, suggesting that endosulfan I and sulfate are developmentally neurotoxic. The waterborne exposure EC 50 s for inhibition of touch response for endosulfan I and endosulfan sulfate were 2.2 μg/L and 23 μg/L, respectively. The endosulfans were highly concentrated by the organisms, and the inhibition of touch response tissue EC 50 , determined from the measured tissue concentrations, was 367 ng/g for endosulfan I and 4552 ng/g for endosulfan sulfate.

Mercury pollution was compared in two Oregon reservoirs of similar size and age, located within the same ecoregion. Cottage Grove Reservoir was distinguished by a history of mercury mining and processing within its watershed, while Dorena Reservoir was not. Mercury concentrations in sediments of the reservoirs, tributary streams, and three species of fish were measured. Sediment mercury concentrations in the main tributary of Cottage Grove Reservoir, which drains the subbasin where past mercury mining occurred, was tenfold higher than mercury in sediments from other reservoir tributaries. There were no significant differences between sediment mercury concentrations in the tributaries of Dorena Reservoir. The average mercury concentration in the basin sediment of Cottage Grove Reservoir (0.67 +/- 0.05 microg/g dry wt) was higher than for Dorena Reservoir (0.12 +/- 0.01 microg/g dry wt). At Cottage Grove Reservoir, maximum mercury concentrations were near or exceeded 1 microg/g wet wt for largemouth bass (Micropterus salmonides) and bluegill (Lepomis macrochirus) epaxial muscle. Muscle mercury concentrations in largemouth bass and crappie (Pomoxis nigromaculatus) from Cottage Grove Reservoir were significantly higher than from the same species from Dorena Reservoir. Numbers of bluegill of the same age available from both reservoirs were too small for statistical comparisons. Mercury concentrations in largemouth bass muscle fluctuated annually in both reservoirs. Fish ages were consistently positively correlated with muscle mercury concentrations in only the point-source-impacted reservoir. These results indicated that a point source, Black Butte Mine, contributed amounts of mercury greatly in excess of mobilization from natural deposits, atmospheric deposition, and small-scale uses of the metal as an amalgamating agent in gold mining.