A multi-mineral natural product inhibits liver tumor formation in C57BL/6 mice

Abstract

C57BL/6 mice were maintained for up to 18 months on high-fat and low-fat diets with or without a multi-mineral supplement derived from the skeletal remains of the red marine algae Lithothamnion calcareum. Numerous grossly observable liver masses were visible in animals on the "western-style" high-fat diet sacrificed at 12 and 18 months. The majority of the masses were in male mice (20 out of 100 males versus 3 out of 100 females; p = 0.0002). There were more liver masses in animals on the high-fat diet than on the low-fat diet (15 out of 50 on high-fat versus 5 out of 50 on low-fat; p = 0.0254). The multi-mineral supplement reduced the number of liver masses in mice on both diets (3 out of 25 male mice in the low-fat diet group without the supplement versus 1 out of 25 mice with supplement; 12 of 25 male mice in the high-fat diet group without the supplement versus 3 of 25 mice with supplement [p = 0.0129]). Histological evaluation revealed a total of 17 neoplastic lesions (9 adenomas and 8 hepatocellular carcinomas), and 18 pre-neoplastic lesions. Out of eight hepatocellular carcinomas, seven were found in unsupplemented diet groups. Steatosis was widely observed in livers with and without grossly observable masses, but the multi-mineral supplement had no effect on the incidence of steatosis or its severity. Taken together, these findings suggest that a multi-mineral-rich natural product can protect mice against neoplastic and pre-neoplastic proliferative liver lesions that may develop in the face of steatosis.

Figures

Figure 1. In situ appearance of livers in a male and female mouse on the HFWD for 18 months

In the male mouse (left), a large liver mass is visible (arrow). In the female mouse, the liver is normal in appearance (arrow). A large amount of fat in the peritoneal wall of the female mouse can be seen, but this is not seen in the male mouse.

Figure 2. Distribution of liver masses in male mice by diet group

Values shown represent the number of male mice at 12 and 18 months (combined) with overt liver masses at necropsy. Differences in the incidence of liver mass formation was assessed for statistical significance using the Fisher Exact Test (two tailed). Differences between high-fat and AIN 76A (low-fat) were significant at p=0.0254; differences between unsupplemented (high-fat alone) and mineral-supplemented (high-fat) were significant at p=0.0129; differences between unsupplemented and mineral-supplemented (high-fat and low-fat combined) were significant at p=0.0054.

Figure 3. Representative histological sections of liver in male mice

A: Normal liver appearance from a male mouse on the mineral-supplemented high-fat diet at 18 months; B: Steatotic liver from a male mouse on the mineral-supplemented high-fat diet at 18 months. Mineral supplementation did not affect the number of mice with steatosis. C and E: Hepatic adenoma (HA) from a male mouse on the high-fat diet without supplementation at 18 months. Arrow indicates compression of adjacent parenchyma. D and F: Hepatocellular carcinoma (HCC) in a male mouse on the high-fat diet without supplementation at 18 months. Hepatocytes form sheets and thick trabeculae without normal architectural features. Fat accumulation (steatosis, S) and necrosis (N) are evident in some areas. Neoplastic (and pre-neoplastic) lesions were more frequent in non-supplemented male mice. All sections are hematoxylin and eosin. (Bars=100 μm).