The use of isolated human lymphocytes in mycotoxin cytotoxicity testing

Abstract

The cytotoxicity of selected mycotoxins against isolated human lymphocytes was investigated, as a means of detecting mycotoxins in extracts derived from cereal samples. The methodology was based on the ability of viable cells to reduce methyl tetrazolium bromide to a purple formazan dye that could be quantitated by spectrophometric means and hence give a measure of the cytotoxicity of added substances. The results showed that there was good correlation with the occurrence of identified mycotoxins with only a minimum of false positives. For example, of the 13 samples of barley or barley derivatives that were positive for the mycotoxins, fumonisin B(1) (FB1) deoxynivalenol (DON) and ochratoxin A (OTA), all gave positive cytotoxicity responses. Two samples negative for mycotoxins gave no cytotoxicity responses. There was little variation between the results for lymphocytes drawn from the same healthy volunteer on three different occasions. Furthermore, for two of the mycotoxins tested (FB1 and DON) it was possible to correlate general levels of mycotoxins present to the cytotoxic response of the lymphocytes but not for OTA, where it was concluded that interfering substances prevented direct correlation. It was concluded that this method was suited for general application as it could handle relatively high number of samples in a short period of time.

Keywords: Cytotoxicity; lymphocytes; mycotoxicosis; mycotoxin screening; mycotoxins.

Figure 1

Dose- response linear regression curve of fumonisin B₁ standard on the human lymphocyte cell line (r = 0.958). All plotted dots of the curves represent means of 3 measurements in 3 distinct test procedures.

Figure 2

Dose-response linear regression curve of deoxynivalenol on the human lymphocyte cell line (r = 0.713). All plotted dots of the curves represent means of 3 measurements in 3 distinct test procedures.

Figure 3

Dose- response linear regression curve of ochratoxin A standard on the human lymphocyte cell line (r = 0.819). All plotted dots of the curves represent means of 3 measurements in 3 distinct test procedures.

Figure 4

Dose- response linear regression curve of neutral barley fraction (B4) on the human lymphocyte cell line (r = 0.811). All plotted dots of the curves represent means of 2 measurements in 3 distinct test procedures.

Figure 5

Dose- response linear regression curve of acid barley fraction (B4 Concentration = ochratoxin A equivalent from instrumental analysis)) on the human lymphocyte cell line (r = 0.714). All plotted dots on the curve represent means of 3 measurements in 3 distinct test procedures.

Figure 6A

Linear regression curve of fumonisin B₁ actual and estimated concentration values (r = 0.946).

Figure 6B

Linear regression curve of deoxynivalenol, actual and estimated concentration values (r = 0.802).

Figure 6C

Linear regression curve of Ochratoxin, actual and estimated concentration values (r = 0.371).