Mechanism underlying carbon tetrachloride-inhibited protein synthesis in liver

Abstract

Aim: To study the mechanism underlying carbon tetrachloride (CCl(4)) -induced alterations of protein synthesis in liver.

Methods: Male Sprague-Dawley rats were given CCl(4) (1 mL/100 g body weight) and (3)H-leucine incorporation. Malondialdehyde (MDA) level in the liver, in vitro response of hepatocyte nuclei nucleotide triphosphatase (NTPase) to free radicals, and nuclear export of total mRNA with 3'-poly A(+) were measured respectively. Survival response of HepG2 cells to CCl(4) treatment was assessed by methyl thiazolyl tetrazolium. Km and Vmax values of nuclear envelope NTPase activity in liver of rats treated with CCl(4) were assayed by a double-reciprocal plot.

Results: The protein synthesis was inhibited while the MDA level was significantly increased in liver of rats treated with CCl(4). In addition, CCl(4) decreased the NTPase binding capacity of nuclear envelope (Km value) in cultured HepG2 cells. Moreover, in vitro ferrous radicals from Fenton's system suppressed the NTPase activity of liver nuclear envelope in a dose-dependent manner. Down-regulation of the nuclear envelope NTPase activity indicated a lower energy provision for nucleocytoplasmic transport of mRNA molecules, an evidence in CCl(4)-treated HepG2 cells correspondingly supported by the nuclear sequestration of poly (A)(+) mRNA molecules in morphological hybridization research.

Conclusion: Inhibition of mRNA transport, suggestive of decreased NTPase activity of the nuclear envelope, may be involved in carbon tetrachloride-inhibited protein synthesis in liver.

Figure 1

CCl₄ inhibits protein synthesis (A) and increases malondialdehyde production (B) in liver of Sprague-Dawley rats. Data are expressed as mean ± SE. ^aP < 0.05 vs control group; ^bP < 0.01 vs their counterpart control group; ^dP < 0.01 vs CCl₄ treatment group.

Figure 2

CC1₄ inhibits nucleotide triphosphatase affinity in nuclei of HepG2 cells. A: Methyl thiazolyl tetrazolium (MTT) metabolic viability assay at a dose of 10 mmol/L showing the inhibitory effect of CCl₄ on growth of HepG2 cells; B: Substrate-affinity (Km, B2) is decreased without any alterations in the substrate binding sites (Vmax,B1) for adenosine triphosphate (ATP) or guanine triphosphate (GTP) in nuclear nucleotide triphosphatase of HepG2 cells treated with CCl₄ (2 mmol/L); C: Enzyme’ activities of nuclei extracts and homogenates from HepG2 cells in control and CCl₄ treatment groups. ^aP < 0.05 vs control group, or nuclear envelop group, respectively; ^bP < 0.01 vs their counterpart control group, or nuclear envelop group, respectively. LDH: Lactose dehydrogenase.

Figure 3

Involvement of reactive radicals in decreased nucleotide triphosphatase activity of nuclei. Data are represented as mean ± SE. ^bP < 0.01 vs H₂O₂/Fe²⁺ group [(0 μmol/L)/(0 μmol/L)]. NTPase: Nucleotide triphosphatase.

Figure 4

CCl₄ decreases total mRNA nuclear export of HepG2 cells. DAPI: 4,6-diamidino-2-phenylindole dihydrochloride; FITC: Fluorescein isothiocyanate.