Mice deficient in glutathione transferase zeta/maleylacetoacetate isomerase exhibit a range of pathological changes and elevated expression of alpha, mu, and pi class glutathione transferases

Abstract

Glutathione transferase zeta (GSTZ1-1) is the major enzyme that catalyzes the metabolism of alpha-halo acids such as dichloroacetic acid, a carcinogenic contaminant of chlorinated water. GSTZ1-1 is identical with maleylacetoacetate isomerase, which catalyzes the penultimate step in the catabolic pathways for phenylalanine and tyrosine. In this study we have deleted the Gstz1 gene in BALB/c mice and characterized their phenotype. Gstz1(-/-) mice do not have demonstrable activity with maleylacetone and alpha-halo acid substrates, and other GSTs do not compensate for the loss of this enzyme. When fed a standard diet, the GSTZ1-1-deficient mice showed enlarged liver and kidneys as well as splenic atrophy. Light and electron microscopic examination revealed multifocal hepatitis and ultrastructural changes in the kidney. The addition of 3% (w/v) phenylalanine to the drinking water was lethal for young mice (<28 days old) and caused liver necrosis, macrovesicular steatosis, splenic atrophy, and a significant loss of circulating leukocytes in older surviving mice. GSTZ1-1-deficient mice showed constitutive induction of alpha, mu, and pi class GSTs as well as NAD(P)H:quinone oxidoreductase 1. The overall response is consistent with the chronic accumulation of a toxic metabolite(s). We detected the accumulation of succinylacetone in the serum of deficient mice but cannot exclude the possibility that maleylacetoacetate and maleylacetone may also accumulate.

Figure 1

The catabolic pathway of phenylalanine and tyrosine. The enzymes referred to in the text are indicated in italics. The dotted lines indicate poorly understood steps.

Figure 2

The organization of the BALB/c mouse GSTZ1/MAAI gene and the DNA constructs made to inactivate the gene in ES cells. The position of primers listed in Table 1 are shown in D.

Figure 3

The expression of GSTZ1 in different mouse tissues. A: Northern blot of mRNA hybridized with a GSTZ1 cDNA (top) and β-actin cDNA (bottom). B: Western blot of liver and kidney cytosolic proteins from male (M) and female (F) wild-type (Gstz1^+/+) and deficient (Gstz1^−/−) BALB/c mice probed with antiserum to recombinant human GSTZ1-1.

Figure 4

Activity of GSTZ1/MAAI in liver and kidney cytosol. Males are shown in the filled columns and females in the open columns. The values are shown as means ± SE, n = 5 to 10 mice. *, P < 0.01 compared with the wild-type (+/+) animals. The Gstz1 genotype is given below each column. A: Activity with CFA as the substrate. B: Isomerase activity with maleylacetone as the substrate.

Figure 5

Weight gain of male BALB/c Gstz1^+/+ and Gstz1^−/− mice given distilled water or water containing 3% phenylalanine. The start of phenylalanine treatment at 5 weeks is indicated (↑). □, Gstz1^+/+, n = 14; ▪, Gstz1^+/+ mice given phenylalanine, n = 6; ○, Gstz1^−/−, n = 13; •, Gstz1^−/− mice given phenylalanine, n = 4.

Figure 6

Light microscopy showing liver and heart pathology in Gstz1^−/− mice. A: Wild-type mouse given 3% phenylalanine for 6 weeks shows normal liver morphology. B: Gstz1^−/− mice given water alone that show focal hepatitis (arrows and inset) at 6 weeks. C: Gstz1^−/− mice given 3% phenylalanine show massive hepatic necrosis (N). D: Gstz1^−/− mice given 3% phenylalanine show macrovesicular steatosis (black arrows). The red infiltrate is red blood cells. E and F: Thrombus and myocardial calcification observed in Gstz1^−/− mice. E: Black arrows show calcification. F: Thrombi in the right atrium (RA) and right ventricle (RV). Original magnifications: ×200 (A, B); ×1000 (D, inset in B); ×100 (C); ×25 (E, F); ×400 (inset in E).

Figure 7

Ultrastructure of livers from two Gstz1^−/− mice given 3% phenylalanine in their drinking water for 28 days. A: Large pleomorphic mitochondria. Inset: mitochondria from wild-type mouse mitochondria. B: Large pleomorphic mitochondria with a crystalline inclusion mitochondria. Original magnifications, ×11,800.

Figure 8

Ultrastructure of kidneys from mice given 3% phenylalanine in their drinking water for 28 days. A: Regular interdigitation seen in wild-type animals. B: Irregular interdigitation seen in the Gstz1^−/− animals. Arrows indicate some of the more irregular areas. C: Regular length and spaced parallel cristae in the mitochondria of the tubules in a wild-type animal. D: Irregular arrangement of variable length cristae of the tubules in a Gstz1^−/− mouse. Original magnifications, ×8100 (A, B).

Figure 9

Induction of other GSTs in female Gstz1^−/− mice. A: SDS-PAGE of soluble proteins from the liver and kidney of wild-type (Gstz1^+/+) and deficient (Gstz1^−/−) mice. The arrow indicates the elevated expression of proteins in the liver of GSTZ1-deficient mice. B: Western blots of soluble liver proteins probed with antiserum to hGSTM1-1, hGSTZ1-1, hGSTP1-1, mGSTA1/2, and ratNQO1. The mouse genotype is shown below each track. Each track was loaded with the same amount of total protein.