*3.3. Functional Consequences of Compromised Zonation: Adaptation to Hepatotoxicants*

Fibrosis associated disturbed lobular zonation causes several adverse functional consequences. An example is compromised ammonia detoxification. The loss of the fine-tuned interaction of the periportal high capacity (urea cycle) and the pericentral high-affinity (GS) compartments leads to increased ammonia blood concentrations. We compared ammonia concentrations in the portal vein, liver vein and heart blood of mice after 1 year of treatment with CCl4 and untreated controls. The much higher concentrations in the hepatic vein of CCl4 mice demonstrate the loss of the capacity of the fibrotic liver to reduce ammonia to very low concentrations of <30 μg/dL (Figure 8).

**Figure 8.** Increased ammonia blood concentrations after 1 year CCl4 treatment. The data are means ± standard errors of 6 mice per group. \*\* *p* < 0.01; \*\*\* *p* < 0.001 compared to the corresponding controls (0).

Moreover, we wondered if the loss of the pericentrally located cytochrome P450 enzymes may lead to resistance against hepatotoxicants that require metabolic activation by these enzymes. To test this hypothesis, we exposed one year CCl4-treated fibrotic mice and olive oil controls to a hepatotoxic dose of APAP (200 mg/kg) (Figure 9A). In oil controls, APAP macroscopically caused the characteristic dotted pattern, indicating pericentral necrosis (Figure 9B) that was confirmed histologically (Figure 9D). Interestingly, APAP-induced no visible necrosis in the fibrotic livers after one year of chronic CCl4 intoxication (Figure 9B,D, Figure S4). These results were confirmed by analysis of the liver enzymes ALT and AST that increased after APAP administration in oil controls but not in fibrotic livers (Figure 9C). Immunostaining for CYP2E1 illustrated that the CYP2E1 positive pericentral regions were destroyed by APAP (Figure 9E). In contrast, the periportalized fibrotic livers without detectable CYP2E1 expression did not show any necrotic hepatocytes around central veins (Figure 9E). The corresponding H&E-stained whole slide scans are available in Figure S4).

**Figure 9.** Acetaminophen (APAP) resistance of mice after one year of treatment with CCl4. (**A**) Experimental schedule. (**B**) Macroscopic appearance. (**C**) The concentration of liver enzymes in the blood. The data are means ± standard errors of 5 mice per time point. \*\*\* *p* < 0.001 compared to the corresponding controls without APAP intoxication. (**D**) H&E-stained tissue. Scale bars: 100 μm. (**E**) CYP2E1 immunostaining. Scale bars: 100 μm.

To analyze whether fibrosis-associated resistance to APAP is a generalizable phenomenon, a similar experiment was performed in mice with BDL-associated fibrosis. For this purpose, BDL mice and sham controls were overnight fasted from the evening of day 20 after surgery and 200 mg/kg APAP were administered on the morning of day 21 to be analyzed 24 h later (Figure 10A). Interestingly, BDL mice were resistant to APAP (Figure 10C,D; Figure S5), similar to the observation made in CCl4-induced fibrosis.

**Figure 10.** APAP resistance of mice 21 days after BDL. (**A**) Experimental schedule. (**B**) Macroscopic appearance. (**C**) H&E staining. Scale bars: 100 μm. (**D**) CYP2E1 immunostaining. Scale bars: 100 μm.
