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.

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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.