Ochratoxin A: in utero exposure in mice induces adducts in testicular DNA

Abstract

Ochratoxin A (OTA) is a nephrotoxin and carcinogen that is associated with Balkan endemic nephropathy and urinary tract tumors. OTA crosses the placenta and causes adducts in the liver and kidney DNA of newborns. Because the testis and kidney develop from the same embryonic tissue, we reasoned that OTA also may cause adducts transplacentally in the testis. We tested the hypothesis that acute exposure to OTA, via food and via exposure in utero, causes adducts in testicular DNA and that these lesions are identical to those that can be produced in the kidney and testis by the consumption of OTA. Adult mice received a single dose of OTA (from 0–1,056 µg/kg) by gavage. Pregnant mice received a single i.p. injection of OTA (2.5 mg/kg) at gestation day 17. DNA adducts were determined by ³²P-postlabeling. Gavage-fed animals sacrificed after 48 hours accumulated OTA in kidney and testis and showed DNA adducts in kidney and testis. Some OTA metabolites isolated from the tissues were similar in both organs (kidney and testis). The litters of mice exposed prenatally to OTA showed no signs of overt toxicity. However, newborn and 1-month old males had DNA adducts in kidney and testis that were chromatographically similar to DNA adducts observed in the kidney and testis of gavage-fed adults. One adduct was identified previously as C8-dG-OTA adduct by LC MS/MS. No adducts were observed in males from dams not exposed to OTA. Our findings that in utero exposure to OTA causes adducts in the testicular DNA of male offspring support a possible role for OTA in testicular cancer.

Keywords: DNA adduct; epidemiology; ochratoxin; testicular cancer; transplacental contamination.

Figure 1

DNA adduct pattern in tissues of mice treated with Ochratoxin A. (A) kidney DNA from control mice; (B) testis DNA from control mice; (C) kidney DNA adduct from mice treated with 1056 mg/kg b.w.; (D) testis DNA adduct from mice treated with 1,056 mg/kg b.w.; (E) Scheme of numbering of the individual DNA adducts. Exposure time of the film was 48 h.

Figure 2

DNA adduct pattern in testis of male mice exposed to ochratoxin A. (A) acute exposure (gavage); (B) mice were fed 4 weeks with feed containing OTA. Doses are expressed as µg/kg b.w. The films were exposed for 24 h panel (A) and 48 h panel (B).

Figure 3

Amount of C-C8dG OTA, expressed as relative adduct level, in organs of mice exposed to increasing amount of ochratoxin A (single dose). Amount in testis = hatched bar; amount in kidney = white bar.

Figure 4

OTA metabolites detected in urine (A) and liver (B) of OTA treated males OTHQ (quinone OTA); GSH conjugated to glutathione; NAC conjugated to N-acetylcystein, DC OTHQ decarboxylated OTHQ; OP OA open ring OTA; OTB dechlorinated OTA; OTC ethylated OTA.

Figure 5

Comparison of litter size, survival and weight of mice treated or not by OTA (CON = control animal; OTA = ochratoxin A treated animal).

Figure 6

DNA adduct pattern of testis of one-month old mice. (A) litter from untreated mother mice; (B, C, D) litter from OTA-treated mother (arrow points C-C8dG OTA); (B & C) correspond to two animals from Mother Number 2; (D) corresponds to one animal from Mother Number 1.

Figure 7

Comigration of mouse testis DNA with kidney DNA or C-C8 dG OTA standard (A) D1 migration; The upper part is cut out and transferred onto new plates as described in Mantle et al., 2010 [30]; (B) C-C8dG OTA spot; Testis spot; comigration ½ C-C8dGOTA + ½ Testis; comigration ½ kidney + ½ testis.

Figure 8

Structure of C-C8 dG-OTA.