PMC

Ethanol-induced UPR in the liver precedes steatosis. (A) qPCR on cDNA prepared from pools of livers dissected from larvae exposed to 350 mM ethanol at 96–128 hpf. Fold changes were calculated by normalizing the comparative threshold (C_T) values calculated as 2^{−CT(target)}/2^−CT(rpp0) to the ones obtained from 0 mM. ***P<0.001 and *P<0.05 by one-way ANOVA and Tukey’s post-hoc test. (B) Fold change in the percent of spliced xbp1 from the total xbp1 message present in liver cDNA from larvae exposed to 350 mM ethanol versus untreated controls, based on the PCR shown in supplementary material Fig. S2A. (C) Fold change in Bip protein levels and Eif2α phosphorylation normalized to β-actin was determined from the immunoblots in supplementary material Fig. S2B. (D) Representative images of whole-mount oil red O staining in larvae exposed to 0 or 350 mM ethanol at the indicated times. The livers are circled. The 32-hour image is enlarged to illustrate the lipid droplets used to score steatosis. Scale bar: 0.2 mm. (E) Average percent of steatosis across 4–8 clutches of larvae exposed to 0 (–) or 350 (+) mM ethanol. ***P<0.0001 by Fisher’s exact test. (F) Average triglyceride (TG) levels (nmol) in livers of larvae exposed to 0 or 350 mM ethanol for 24 hours were normalized to total protein (μg). Mean ± s.e.m. n=4 clutches, *P<0.05 by a Student’s t-test.

Ethanol metabolism and ROS are required for secretory pathway stress. (A) Heatmap of qPCR data from liver cDNA measuring the expression of genes involved in the UPR. The fold changes of the median C_T values are in supplementary material Table S3. All six clutches are aligned according to the order of the lowest to the highest expression of bip in 350 mM ethanol treatment. (B) Representative images of the tail of Tg(l-fabp:Dbp-EGFP) 120 hpf larvae treated with 0 or 350 mM ethanol or co-treated with AA and 350 mM ethanol for 24 hours. The rectangles in the left panel are magnified in the right panel. Scale bars: 0.2 mm for the left panel and 0.04 mm for the right panel. Note that the curved tail in untreated larvae is an artifact of fixation. (C) Immunoblots of transgenic Tg(l-fabp:Dbp-EGFP) larvae treated as in B using anti-GFP antibody and a non-specific band serving as a loading control. (D,E) Low concentration of ethanol and H₂O₂ synergize to induce UPR (D) and oxidative stress (E). Heatmaps of qPCR data from the livers of larvae exposed to individual treatments of 100 mM ethanol or 2.1 mM H₂O₂, or a co-treatment of the two, for 24 hours. The individual C_T values are in supplementary material Tables S4 and S5 for D and E, respectively. All six clutches are aligned according to the order of the lowest to the highest expression of bip (D) or sod2 (E) in the co-treatment of 350 mM ethanol and 2.1 mM H₂O₂.

Ethanol is rapidly internalized, utilized and metabolized in zebrafish larvae. (A) Internal ethanol concentration was measured in homogenates of whole larvae treated with the indicated ethanol concentrations at 96–128 hpf. Values are in mM; mean ± s.e.m. n=6 clutches, n=260 larvae. All concentration points are statistically significant (***P<0.001) from the controls. Samples labeled as n.s. did not differ from one another. (B) Internal ethanol concentration determined from whole larvae treated with 350 mM ethanol for the indicated durations; mean ± s.e.m. n=6 clutches, n=200 larvae. All time points on the curve are significantly different (P<0.001) from t=0. ***P<0.001 versus t=32 hours. (C) Ethanol internalization and consumption was measured by calculating the external (water) ethanol concentration at t=0 and t=32 hours of larval exposure. To account for ethanol evaporation, concentration was also calculated from media that lacked larvae and was maintained in parallel. Values are the average of triplicate measurements of samples obtained from four clutches (mean ± s.e.m.); ***P<0.001. (D) The effects of CMZ and 4MP on ethanol utilization was measured in naïve larvae (black bars) or larvae pre-treated with 100 μM CMZ (light gray) or 1 mM 4MP (dark gray) at 94 hpf and then co-exposed to 350 mM ethanol from 96 to 128 hpf at a density of 1 larva/ml. Dashed line marks 350 mM ethanol. Values were calculated from triplicate measurements on three clutches (mean ± s.e.m.). ***P<0.001. (E) Untreated larvae (N/T) and larvae pre-treated with 100 μM CMZ, 1 mM 4MP, 3 mM CYA or 40 mM OAc at 94 hpf and then co-exposed with one of these drugs and 350 mM ethanol from 96 to 120 hpf were scored for mild or severe phenotypes, as shown in the representative images in the panels on the right. The severely affected phenotype (gray bars) was significantly reduced in larvae co-treated with CMZ or 4MP, but increased with CYA. *P<0.05 and **P<0.01. Statistics in all panels were calculated by one-way ANOVA and Tukey’s post-hoc test.

Concentrations of ethanol exceeding 350 mM reduce survival and induce multisystemic morphological abnormalities in 4 dpf larvae. (A) Larvae at 96 hpf were exposed to 0 mM, 87.5 mM (0.5%), 175 mM (1.0%), 262.5 mM (1.5%), 350 mM (2.0%), 437.5 mM (2.5%) and 525 mM (3.0%) ethanol and scored for viability at 128 hpf; mean ± s.e.m. n=6 clutches, n=125 larvae per treatment; ***P<0.001 versus 0 mM. The dashed line indicates the optimal concentration. (B) Kaplan-Meier survival curve of larvae exposed to 0 mM or 350 mM ethanol for 32 hours and scored for survival at every 4 hours; n=13 clutches, n=562 larvae per cohort. The P-value is indicated as determined by log-rank test; ***P<0.001 versus 96 hpf in 350 mM curve. (C) Images of one Tg(fabp10:dsRed) larva during exposure to 0 mM ethanol and another during exposure to 350 mM ethanol from 96 to 128 hpf. Arrowhead indicates lordosis at 104 hpf; arrows indicate hepatomegaly at 104 hpf and pericardial edema at 108 hpf. Scale bars: 1 mm in the upper panels and 0.2 mm in the lower panels. (D) Lordosis and edema were scored in larvae that survived 32-hour exposure to ethanol at concentrations of 0–525 mM; mean ± s.e.m. n=6 clutches, n=100 larvae. Except for the 87.5 mM ethanol group, all concentration points on both curves are significantly different (***P<0.001) compared with 0 mM. There is no significant (n.s.) difference in the percent of lordosis and edema in larvae treated with 350 mM ethanol or greater. (E) Morphological changes during 32 hours of exposure to 350 mM ethanol were averaged from ten clutches (n=442 per group). The percent of unaffected larvae was significantly reduced at all time points starting at 8 hours of exposure; the percent of larvae with lordosis alone was significantly increased from 8 hours of exposure; and the percent of larvae with both lordosis and edema was significantly increased at 12 hours of exposure and later; P<0.001. Untreated larvae (n=442) scored in parallel did not display any of these phenotypes at any time points (not shown). All statistical significance, except where indicated, was calculated by one-way ANOVA and Tukey’s post-hoc test.

Ethanol-induced ROS production and morphological abnormalities in zebrafish are rescued by antioxidants and inhibitors of ethanol metabolism. (A) ROS production was measured by assaying CM-H₂DCFDA fluorescence in the media during exposure to 350 mM ethanol. The arbitrary units of fluorescence measured in duplicate from larvae treated with 350 mM ethanol were normalized to corresponding untreated fish and the average fold changes of four clutches are shown. *P<0.05 as determined by a one-sample Student’s t-test. (B) Larvae were either pre-treated with 125 μM AA, 20 μM NAC or 100 μM CMZ at 94 hpf or injected with 4–6 nl of 0.1 mM cyp2y3 morpholino at 0 hpf and then exposed to 350 mM ethanol at 96 hpf for 24 hours. **P<0.01 by one-way ANOVA and Tukey’s post-hoc test. (C) A heatmap of relative expression based on qPCR from cDNA isolated from the livers of larvae exposed to 350 mM ethanol alone or co-treated with CMZ, 4MP, AA or NAC for 24 hours. Each row is a gene and each column is a single clutch, and the color range (red – high, blue – low) was determined via the median method in GENE-E. CMZ, 4MP, AA and NAC treatments alone did not affect the expression of these genes when compared with untreated larvae (0 mM) and thus are not shown. The fold changes of the median C_T values of six clutches are shown to the right and individual C_T values are in supplementary material Table S2. The median was calculated for each row (gene) and subtracted from each data point. All six clutches are aligned according to the order of the lowest (blue) to the highest (red) expression of sod2 in 350 mM ethanol treatment. (D) Representative images of unaffected, and mildly or severely affected larvae are shown on the right. The phenotypes were scored in an average of nine clutches (n=210 larvae per cohort). ***P<0.001 refers to severely affected fish (gray bars) and was calculated by one-way ANOVA and Tukey’s post-hoc test.

Ethanol metabolism and ROS are required for steatosis and HSC activation. (A) Quantification of whole-mount oil red O staining in 120 hpf larvae that were either untreated/uninjected (N/T), or injected with 4–6 nl of 0.1 mM cyp2y3 morpholino solution at the one- to four-cell stage (0 hpf), or pre-treated with 100 μM CMZ, 1 mM 4MP, 125 μM AA, 20 μM NAC at 94 hpf and then exposed to 0 or 350 mM ethanol at 96 hpf for 24 hours. Note that, although each of these treatments did not affect the spontaneous steatosis levels, they significantly reversed the rate of ethanol-induced steatosis. ***P<0.0001 by Fisher’s exact test. (B,C) qPCR analysis of Srebp1 (B) and Srebp2 (C) target genes in cDNA isolated from the livers of untreated larvae or larvae exposed to 350 mM ethanol alone or co-treated with CMZ, 4MP, AA or NAC for 24 hours from six different clutches. Data are represented as mean fold changes to untreated samples with s.e.m. (D) Ethanol-induced HSC activation is partially rescued by CMZ. The percent of larvae with an altered HSC phenotype is an indication of activation, including laminin secretion (i.e. altered morphology + laminin). Over 70% of untreated control and CMZ-treated larvae have a normal HSC morphology with complex processes and no laminin deposition in the liver, whereas only 11% of larvae have some HSCs with a normal phenotype. Co-treatment of CMZ and ethanol increases the percent of larvae with normal HSCs to 42%. The number of larvae analyzed for each condition is indicated in parenthesis on top of each bar. (E) Working model illustrating that ethanol can generate ROS either through Cyp2-mediated metabolism (via acetaldehyde) or through altering mitochondrial metabolism and that high ROS levels directly cause protein damage. Together, these lead to unfolded protein accumulation in the ER, UPR induction and, by an as-yet-unknown mechanism, steatosis.