The effect of butylated hydroxyanisole and butylated hydroxytoluene on behavioral development of mice. Stokes JD, Scudder CL, Dev Psychobiol 1974 Jul;7(4):343-50


From full text of study

John D. Stokes
Charles L. Scudder
Institute for the Study of Mind, Drugs and Behavior
Department of Pharmacology
Stritch School of Medicine
Loyola University
Maywood, Illinois

Abstract

The chronic ingestion of .5% butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) by pregnant mice and their offspring resulted in a variety of behavioral changes. Compared to controls, BHA-treated offspring showed increased exploration, decreased sleeping, decreased self-grooming, slower learning, and a decreased orientation reflex. BHT-treated offspring showed decreased sleeping, increased social and isolation-induced aggression, and a severe deficit in learning.
Introduction

For over 20 years the antioxidant-type food preservatives, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been widely used throughout both the United States and other parts of the world. Although only a few definite toxicological changes have been clearly correlated with these preservatives (e.g., interalveolar septal thickening and generalized lung tissue disorganization upon intraperitoneal injection of BHT [Marino & Mitchell, 1972], doubt has been expressed as to their safety (Anonymous, 1965). Of the many investigations concerning BHT, at least 1 study reported a teratogenic effect - anopthalmia (Brown, Johnson, & O'Halloran, 1959). However, this aberration was not found on subsequent attempts at replication (Anonymous, 1965). Other effects more commonly associated with these phenolic antioxidants are depressed growth rate (Brown et al, 1959; Johnson & Hewgill, 1961), increased liver weight (Brown et al, 1959; Gilbert & Goldberg, 1965; Johnson & Hewgill, 1961); and increased serum-cholesterol levels (Johnson & Hewgill, 1961). All of these occur without gross morphological changes to the animal.

In this laboratory we have shown that drugs and other chemicals may alter the behavioral development of an animal without gross morphological changes (Abbatiello & Scudder 1969; Antonita, Scudder, & Karczmar, 1968; Richardson, Karczmar & Scudder, 1972a; Scudder & Richardson 1970; Stokes, Scudder & Karczmar 1972). The purpose of this paper is to communicate the subtle nature of the changes in behavior that occur due to the ingestion of a "safe" food additive. Such behavioral changes may constitute an insidious, overlooked challenge to the developing organism. This paper is not an attempt to challenge the suitability of the antioxidants for man, although some of the guidelines for such a study are met (Weil, 1972), but to emphasize the fact that, at the present time, food additives are not being tested for behavioral or develomental behavioral changes that they may cause (Friedman & Spiher,1971).

Methods

Mated pairs of Swiss-Webster mice (Mus musculus) were separated at random into 3 groups. The 1st group (Controls) received a diet of powdered Purina Rat Chow and Pet Milk. The 2nd and 3rd group received a diet identical to that of the 1st group, but contaminated either with .5% by weight Tenox Food Grade BHA or with .5% by weight Tenox Food Grade BHT (Eastman Organic Chemicals), respectively. . . .

Discussion

These studies have established some behavioral characteristics of mice that were chronically pre- and postnatally treated with BHA or BHT. The BHA-treated mice exhibited increased exploration, decreased sleeping, decreased grooming of self, slow learning and a decreased orientation reflex when compared to the untreated control animals. On the other hand, the BHT-treated animals exhibited decreased sleeping and learning, as well as increased social and isolation-induced aggression. Because the mice were exposed to the antioxidants both pre- and postnatally, we cannot state at present whether the effects were due to BHA and BHT ingested by pregnant mothers, by their pups, or by both. The lack of significant differences between the acutely treated animals in regard to aggression suggests that the effects are developmental.

Behavioral changes such as those demonstrated above commonly occur when the levels of neurotransmitters are altered (Karczmar & Scudder, 1969; Richardson et al., 1971a, b). A pilot study indicated that the chronic BHA and BHT treatment induced changes in the whole brain serotonin and norepinephrine levels and in cholinesterase activity (Stokes et al, 1972). Brain serotonin seems to be involved in slow sleep, as shown by several investigators (Apriso & Hingtgen, 1972), and the decrease in sleep shown at present in our "city" studies may be related to the decrease in serotonin levels which we demonstrated previously (Stokes et al, 1972).

Whether or not these antioxidants have a direct effect on neurohumoral levels or transmission has not been determined as yet. Because these antioxidants are used industrially to prevent the oxidation of fats, we speculate that BHA and BHT are having some effect on membranes which are highly lipid in nature. Cytokinesis or some other aspect of the transmitter storage physiology seems to be the most probable site of action. Possibly, the antioxidants may stabilize the synaptic vesicle membrane, and thereby interfere with release.

In our previous studies, learning and aggression were asociated as a "syndrome;" that is, these behaviors changed in parallel when related mice genera and strains were compared or when various types of drugs were used in a particular strain (Karczmar, Scudder & Richardson, in press; Scudder, Karczmar, Everett, Gibson & Rifkin, 1966). We note that at least BHT had a differential effect on the components of the syndrome.

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