ToxicOlogy Lrftws, 10 (1982) 249-253

Elsevier Biomedical Press 249

IN VIVO AND IN VITRO STWDfES ON THE BrNDING NATURE OF TERREIC AC3D WITH MACROMOLECULES SUCH AS PROTEIN AND NUCLEIC ACIDS

T. SUBRAMANIAN*~ K. MOHAN NAMASIVAYAM and E.R.B. SHANMU~ASUNDARAM

University Biochemical Laboratories, University of Mrrdsss, Guindy, Madras-600025 {India)

(Received June 29th, 1981)

(Revision received August 6th, 1981)

(Accepted August I lth, 1981)

SUMMARY

The binding of terreic acid to macromolecules such as nucleic acids and protein was examined. In vivo

studies using [t%]terreic acid showed that radioactivity was incorporated into the protein and nucleic

acid fractions of the liver of mice. In vitro experiments with human serum showed that [t%Z]terreic acid

bound to the albumin component and spectral studies indicated that terreic acid also combined with

hepatic DNA.

INTRODUCTION

Though a number of mycotoxins and their effects on biological systems are known, the mechanism of action for most of the mycotoxins has not been clearly demonstrated. Recent investigations have suggested that ‘mycotoxin-macromo~e- cular binding’ may play a significant role in the toxicity of many mycotoxins, e.g. aflatoxin [6], ochratoxin [2] and citrinin [5]. Such considerations are based on the hypothesis that binding of mycotoxins with macromolecules might alter the secon- dary and tertiary structures of macromolecules and subsequently modify their function. Mycotoxins, e.g. aflatoxins [Ei], luteoskyrin [14], ochratoxin [2], citrinin [5], penieillic acid, and patulin [I] have been reported to bind to macromolecules such as protein and nucleic acids. Therefore, the nature of binding of the myco-

toxin, terreic acid, with serum proteins and nucleic acids was studied.

* Present address: Department of Microbiology, Madurai Medical College, Madurai-625020 (India).

037%4274/82/0000-0000/$02.75 0 Elsevier Biomedical Press

250

MATERIALS AND METHODS

Uniformly labelled [r4C]glucose (5 mCi) was obtained from Bhabha Atomic

Research Centre (Bombay, India).

Preparation of radioactive terreic acid

A strain of Aspergillus terreus, isolated in our laboratory from contaminated

feeds, was grown in 1 1 of minimal medium with [r4C]glucose and other constituents

[l 11. After 15 days of growth, the radioactive terreic acid (Fig. 1) was isolated from

the cultures and crystallised by the method of Kaplan et al. [7]. The specific activity

of the terreic acid crystals was 4.2. 10’ cpm/g.

In vivo experiment in mice

Male and female albino mice weighing 20-25 g were used. 5 mg radioactive terreic

acid (200000 cpm) dissolved in sterile water was injected i.p. After 8 h the mice were

killed, the livers removed and the lipid, protein and nucleic acid fractions were

obtained as follows.

Liver tissue (0.5 g) was extracted 3 times with 5 ml of a mixture of chloroform and

methanol (2: 1). The extracts were pooled and concentrated to a known volume.

The tissue remaining after extraction of the lipid fraction was homogenised in 5

ml water, 5 ml 10% ice-cold trichloroacetic acid was added and left for 30 min.

The preciptate of protein and nuclei acid was washed 3 times with 10% trichloro-

acetic acid, then 5 ml of 5% trichloroacetic acid was added and held at 70 “C for 15

min. It was centrifuged and the supernatant representing the nucleic acid fraction

was separated.

The protein precipitate which is the protein fraction was homogenised in 5 ml

water.

0.5 ml of each fraction was spread on a planchette and dried at 60°C. Radio-

activity was measured using a thin end window Geiger Muller tube connected to

Panax-type 1OOC counter. No corrections were necessary for self-absorption since

only a thin film was used. Measured activities were corrected for background

counts. The radioactivity in the various fractions of liver tissue was expressed as

specific activity (cpm/mg of tissue). Experiments were carried out on 10 mice.

0

0 OH

0

CH3 0

Fig. 1. Structure of terreic acid.

251

In vitro binding of terreic acid to serum protein

1 ml of fresh normal human serum was mixed with radioactive terreic acid

(100000 cpm) and incubated at 37°C for 3 h. The incubated material was dialysed

overnight against 1 1 of 0.083 M barbitone buffer (pH 8.6) and the dialysed serum,

50 ~1 subjected to paper electrophoresis in barbitone buffer at 180 V and 5 mA for 4

h. After the run the papers were dried and stained with bromophenol blue.

The individual protein fractions were extracted with 0.1 N sodium hydroxide, a

known volume transferred to planchettes, and counted.

In vitro binding of terreic acid with mouse liver DNA

Liver slices were prepared [9] and incubated with 5 mg of terreic acid in 40 ml 0.2

M phosphate buffer (pH 7.0) at 37°C for 4 h. A control was carried out without

terreic acid. DNA was isolated by the method of Kirby [8], dissolved in water and

the UV absorption maxima read in a Cecil-CE 272 Linear Readout UV Spectro-

photometer.

Binding of terreic acid to pure DNA

DNA (10 mg) isolated from mouse liver was incubated with 2 mg of terreic acid in

10 ml of water at 37 “C for 4 h. After incubation it was dialysed overnight against 2 1

distilled water with 3 changes of water during the period. Pure DNA was treated

similarly without terreic acid to serve as a control. The UV absorption maxima of

the above dialysates were read in a spectrophotometer.

RESULTS AND DISCUSSION

Table I shows the incorporation of [14C]terreic acid into various fractions of

mouse liver. There is no incorporation of radioactivity into the lipid fraction.

However, there is incorporation into the protein and nucleic acid fractions being

greater in the latter. Swenson et al. [12] reported that injection of [3H]aflatoxin Br

TABLE I

[‘4C]TERREIC ACID INCORPORATION IN VARIOUS FRACTIONS OF MICE LIVER

(MEAN + S.D.)

Fractions Spec. act. (cpm/mg of tissue)

Lipid Nil

Nucleic acid 9.2? 1.1

Protein 2.1 f 0.08

Fig. 2. UV absorption maxima of DNA (A) and DNA-terreic acid (B) complex.

into rats yielded covalently bound derivatives in hepatic DNA, RNA and proteins.

This is in accord with our result indicating the interaction of terreic acid with macro-

molecules.

When incubated with human serum in vitro radioactivity was detected only in the

albumin fraction (15.2%).

The in vitro binding effect of terreic acid with purified DNA is shown in Fig. 2.

The absorption maxima of DNA is 260 nm. The absence from the spectrum of a

peak at 260 nm suggests the formation of a complex of terreic acid with DNA.

Similar changes have been described for ochratoxin [2] and other mycotoxins [4,

101.

Attempts to identify the spectral variation in DNA isolated from tissues incubated

with terreic acid have been unsuccessful, probably the complex becomes hydrolysed

during isolation.

Visvanatha Rao et al. 1151 showed that aflatoxin is absorbed from the intestine

and transported as a complex with serum albumin. Terreic acid may be transported

in a similar manner.

Ueno [13] reported that the 12,13-epoxy trichothecenes combined with macro-

molecules and produced disease in a number of animals and man. Chu [3] suggested

a possible mechanism for the binding of aflatoxin Br to macromolecules involving

metabolic activation by microsomal enzymes to an epoxide which reacted subse-

quently with nucleophilic centres in nucleic acid, protein and -SH groups. A similar

mechanism may account for the reaction of the toxin terreic acid with tissue macro-

molecules.

There is ample evidence that many epoxides are carcinogenic and our results

suggest that terreic acid may have a similar action consequent upon its reaction with

DNA and protein.

253

The authors wish to thank Professor (Mrs.) Radha Shanmugasundaram for criti- cal discussions, and the University Grants Commission for the award of two Senior Research Fellowships (to T.S. and K.M.N.)

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