A new mechanism of cancer initiation that involves the transformation of hepatocytes into preneoplastic single hepatocytes and minifoci positive for glutathione S-transferase P-form (GST-P) in rat livers: 3D analysis using a vibratome

Abstract

Background: Cancer initiation has long been "unknowable" in biology and medicine. In 1987, however, Moore and our research group observed single hepatocytes and minifoci that were strongly positive for glutathione S-transferase P-form (GST-P) in the rat liver as early as 2 to 3 days after initiation by diethylnitrosamine prior to the induction of GST-P⁺ foci and nodules. The induction of GST-P⁺ single hepatocytes, precursors of GST-P⁺ foci and nodules, was considered genetic. But, the details of the induction mechanism have remained unclear despite various examinations over a long period.

Methods: Male Sprague-Dawley rats (aged 6 weeks) were fed a basal diet containing either benzyl isothiocyanate (BITC, 0.5% by wt) or 2-acetylaminofluorene (AAF, 0.04%) ad libitum for appropriate time intervals. All animals were anesthetized and euthanized. The livers obtained were excised, cut into 3- to 4-mm-thick slices and fixed in cold acetone at 4 °C. The liver specimens were then sliced into 25-µm-thick sections in PBS using an automated microtome (Vibratome 1500 Sectioning System, Vibratome Products, NY, USA). Immunocytochemical staining was performed in free solution, and the results were examined via digital light microscopy (Coolscope, Nikon, Tokyo).

Results: 3D analysis using a vibratome showed that GST-P is rapidly excreted into the bile of the liver of animals in response to strong carcinogenic stress caused by promoters or initiators. "Rapid biliary excretion of GST-P" was widely and commonly observed in all hepatocytes, GST-P⁺ single hepatocytes, minifoci, foci and nodules under appropriate conditions. Surprisingly, on the basis of these key findings, a new mechanism of cancer initiation involving the transformation of hepatocytes into GST-P⁺ single hepatocytes and minifoci in animal livers was identified. In addition, the initiation process was determined to be nongenetic because mutation is an invisible rare event.

Conclusions: This short review describes several details about breakthrough findings on cancer initiation in rat livers, the application of 3D analysis to other cancers and the importance in the genetic analysis in malignant diseases.

Keywords: 3D analysis; GST‐P; cancer initiation; chemical hepatocarcinogenesis; tumor markers; vibratome.

FIGURE 1

Induction of GST‐P⁺ preneoplastic cell populations in rat livers. (A) Solt–Farber protocol for the induction of preneoplastic cell populations. DEN, injection of diethylnitrosamine in a single i.p. injection at a dose of 200 mg/kg; PH, 2/3 partial hepatectomy; B.D., basal diet; AAF, basal diet containing AAF (0.02%). (B) Immunocytochemical staining patterns of (a) GST‐P⁺ single cells and minifoci induced 2 days after DEN administration and (b) GST‐P⁺ foci and nodules induced after 5 weeks. a), b) were stained with microtome‐ and vibratome‐prepared liver specimens, respectively. CV represents the central vein. Bb was reprinted from Ref. [22] with permission from Wiley.

FIGURE 2

Initiated cell theory for the induction of GST‐P⁺ single hepatocytes according to Farber et al., In this figure, = represents DNA in the nucleus of a hepatocyte, and the mutation is shown by x.

FIGURE 3

3D analysis of GST‐P⁺ single hepatocytes. (A) Representative staining pattern of GST‐P⁺ single hepatocytes induced in the livers of rats fed a basal diet containing AAF (0.02%) and NAC (0.5%) for 4 weeks. White arrowheads represent staining of canaliculi; (B–E) examples of GST‐P⁺ single hepatocytes positive for the excretory portions (arrowheads). Reprinted from Ref. [17] with permission from Elsevier.

FIGURE 4

3D analysis of canalicular networks and bile ducts in BITC‐treated rat livers with a GST‐P antibody. (A) and (B) Staining patterns of the livers of rats fed the BITC‐containing diet for 5 and 10 days, respectively. (A') Higher magnification of (A). (C) Staining pattern of the nonfed control. BD, interlobular bile duct; CV, central vein. Reprinted from Ref. [25] with permission from Elsevier.

FIGURE 5

3D analysis of preneoplastic cell populations induced in rat livers by AAF. The animals were given dietary AAF (0.04%) for 7 weeks. (A–E), Single hepatocytes and minifoci induced after 2 weeks; (F, G), minifoci induced after 4 weeks; (H–J), minifoci and foci induced after 5 weeks; (K), a nodule induced after 7 weeks. Arrows, GST‐P⁺ single hepatocytes; arrowheads, canaliculi linked to GST‐P⁺ single cells; white arrowheads, GST‐P⁺ canaliculi. Reprinted from Ref. [25] with permission from Elsevier.

FIGURE 6

Carcinogenic action of initiator and promoter agents on hepatocytes, which give rise to single GST‐P⁺ hepatocytes and intact hepatocytes, respectively, in rat livers.