Assessment of the role of aflatoxin in the aetiology of carcinoma of the mucosa of the ethmoid

Abstract

The present investigations were planned to assess the role of aflatoxin B1 (AFB1) and /or virus in the aetiology of ethmoid carcinoma using pig as a model in vivo and bovine ethmoid mucosa culture in vitro.

Thirty – two, large white Yorkshire piglings of two – three months age were procured from the University pig Breeding Farm, Mannuthy and divided at random into four groups of eight each.

The pigs in group I and group II were administered aflatoxin B1 (0.070 mg/kg b.wt/inoculums by intravenous route at weekly interval for six months) and /or ethmoid tumour extract (2 ml/pig/inoculums, intranasally, at fortnight interval for three months). The pigs in group III were administered ethmoid tumour extract alone, while the pigs in group IV were kept as negative controls.

During the period of observations of 18 months all the pigs of different groups given AFB1 and /or ethmoid tumour extract appeared healthy and no clinical manifestation of the carcinoma of the mucosa of ethmoid was observed. However, there was appreciable reduction in the weight and mild degree of depression.

In the AFB1 treated pigs, sacrificed at 9, 12, 15 and 18 months of investigation, the ethmoid mucosa had grayish white, soft and oedematous appearance along with scattered small pale elevations at necropsy. Histologically, the ethmoid mucosa exhibited hyperaemia, varying degree of mononuclear cell infiltration and fatty degeneration in the initial stages. In the later stages, there was proliferation of mucous glands showing acinar, tubular or papillary arrangements. Occasionally papillary projection of the surface epithelium and focal squamous metaplasia were also observed. Ultrastructural features of the cells of the ethmoid mucosa consisted of both productive and degenerative changes. The cells had sparse cytoplasmic organelles. The poor cytoplasmic contents and irregular nucleus with nucleolar margination were the other electron microscopic features observed in the ethmoid mucosa of AFB1 treated pigs.

AFB1 in the range of 43.12 – 139.43 ppb could be detected in the blood of 52.37 percent of the ethmoid tumour bearing cattle analysed in the present study.

The blood samples from the AFB1 treated pigs were positive for AFB1 (40-160 ppb) upto 10 days after the withdrawal of treatment whereas AFM1 could be detected in blood sample of one pig only upto 3 days after the treatment. The ethmoid mucosa analysed after 3 months and at subsequent specified intervals was consistently negative for AFB1 and AFM1.

By concerted efforts cells of the mucosa of the ethmoid were established in vitro. AFB1 treatment of long term epithelial cultures intiated from the primary culture of bovine ethmoid mucosa origin resulted in morphological transformation accompanied by increased growth in soft agar and cytochemical positivity of gamma glutamyl transpeptidase. This confirmed the tumourigenicity of AFB1. The xenotransplantation of these in vitro transformed epithelial cells in mice was not successful.

Electron microscopic studies of the cells of the carcinoma of the ethmoid mucosa in spontaneous cases of cattle revealed varying ultrastructural features. The neoplastic cells were either well differentiated secretary structures or undifferentiated ones. Desmosomes and tight junctions were seen between the epithelial cells. Endoplasmic reticulum and mitochondria varied in their contents and degree of disorganization. Nucleus was highly pleomorphic and predominantly euchromatinic.

The retroviral like particles were demonstrated intracellularly and occasionally in extracellular spaces in the neoplastic cells of 7 tumour bearing cattle. Similar particles were also seen in the cell free ethmoid tumour extract in three of21 tissues examined.