15

Mutation Research, 55 (1978) 15--30

© Elsevier/North-Holland Biomedical Press

CYTOTOXIC EFFECTS OF MALEIC HYDRAZIDE

Z. SWIETLII~SKA and J. ZUK

Institute o f Biochemistry and Biophysics, Polish Academy o f Sciences, 02-532 Warsaw,
Rakowiecka 36 (Poland)

(Received 28 February 1978)

(Revision received 7 June 1978)
(Accepted 9 June 1978)

Contents

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Chemical characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Effect on cellular metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Induction of chromosomal aberrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Sensitivity to the toxic effects of MH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Uses in agriculture and the food industry, and the genetic risk to man ........... 25
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Summary

S i n c e 1 9 5 0 , m a l e i c h y d r a z i d e ( M H ) h a s b e e n i n t r o d u c e d i n t o a g r i c u l t u r e as a
m a j o r c o m m e r c i a l h e r b i c i d e a n d a d e p r e s s a n t o f p l a n t g r o w t h i n n u m e r o u s cir-
c u m s t a n c e s s u c h as s u p p r e s s i o n o f s p r o u t i n g o f v e g e t a b l e s a n d s t o r e d f o o d
crops, control of sucker growth on tobacco plants, ratardation of flowering and
p r o l o n g a t i o n o f d o r m a n c y p e r i o d . S i n c e 1 9 5 1 M H h a s b e e n k n o w n as a n effec-
t i v e c h r o m o s o m e - b r e a k i n g a g e n t i n h i g h e r p l a n t s , i n s h a r p c o n t r a s t w i t h its l o w
effect o n t h e c h r o m o s o m e s a n d general h e a l t h of tested m a m m a l s . T h e select-
i v i t y o f a c t i o n o f M H i n p l a n t s a n d a n i m a l s was o b v i o u s l y t h e m a i n r e a s o n o f

Abbrevations: DMBA, 7,12-dimethylbenz[a]anthracene; MH~ maleic hydrazide.

16

low interest devoted to the chemical by people working in the field of envi-
ronmental mutagenesis.
In early works the inhibitory effects of MH on plant growth were mainly
considered to result from the suppression of plant metabolism (i:~hibition of
enzymic activity) and interference of the c o m p o u n d with plant hormones and
growth regulators. More recently, numerous experiments performed with
various plant species have shown that MH acts as an inhibitor of the synthesis
of nucleic acids and proteins. Similar results have been obtained with animal
t u m o u r cells.
The chromosome-breaking effect of MH on plant chromosomes resembles
very closely the chromosome-breaking properties of alkylating agents and other
mutagenic compounds such as mitomycin C. MH-induced chromosomal aberra-
tions have also been recorded in grasshoppers, fish and mice, although tests
with some mammalian cell lines gave negative results.
Among higher plants, selective sensitivity to the toxic effects of MH is well
proved. This p h e n o m e n o n seems to be due to the differential ability of various
plant species to detoxicate the chemical. Plants can break down MH into
several products, one of which, hydrazine, is a well-known mutagen and car-
cinogen. MH does n o t seem to be toxic to bacteria and fungi. The c o m p o u n d is
degraded by soil microflora and hence can be utilized as a source of nitrogen
nutrition.
MH proved to be of low toxicity to mammals, but in some instances it
decreased the fertility of rats. The reported carcinogenic effects of MH in mice
and rats raise the question of its risks to man.

Introduction

SchSne and H o f f m a n discovered in 1949 that maleic hydrazide is a p o t e n t

inhibitor of plant growth. Since then the c o m p o u n d has been widely used in
agriculture as a selective weed killer and as a depressant of plant growth in vari-
ous circumstances. Shortly after introducing MH as a major commercial her-
bicide, Darlington and McLeish [15] discovered its p o t e n t chromosome-
breaking activity in plant cells.
The widespread use of MH in agriculture and the food industry, and its
marked chromosome-breaking properties, caused an urgent need for the testing
of the c o m p o u n d for possible toxic and carcinogenic activity to animals and to
man. The first studies in this respect were undertaken by Salaman and Rao
[96] and Barnes et al. [3]. The experiments with rabbits, rats and mice did n o t
reveal any effects on growth and general health of the treated laboratory
rodents, and accordingly MH was claimed to be harmless for animals.
However, more recent papers produced information on carcinogenic prop-
erties of MH in mice [16,20,22], on induction of chromosomal aberrations in
various groups of animals [55,66,67], and on inhibition of the synthesis of
nucleic acids and protein in Yoshida-Sarcoma Ascites cells [26]. All these
findings again focus our attention on the problem of possible carcinogenicity of
MH to man.
Although much work has been concerned with the cytotoxic effects of MH,
 17

the mechanism of its action in plant and animal cells remains uncertain.
Obviously there still exists a strong need for further testing of the c o m p o u n d
for genetic and environmental toxicity.

Chemical characteristics

Maleic hydrazide (1,2-dihydro-3,6-pyridazinedione) was synthesized by Cur-

tius and Fosterling in 1895 from maleic anhydride and hydrazine. MH is a
white solid, decomposing at 260°C. It is slightly soluble in cold water (0.4% at
20°C) and more soluble in h o t water. Reaction of MH with basic c o m p o u n d s
results in formation of MH salts which are more soluble in water than is the
parent compound. In agriculture, MH is used mainly as its diethanolamine salt
and sodium salt, both easily soluble in water.
There are three possible tautomeric forms of MH, b u t the generally accepted
is form II.

0 OH OH
II t r
HC~c'~NH NC/'c~ N HC/C~N
II I II I II I
HC~.C/NH HC~.c/NH HC~.c//N
II II I
0 0 OH
I lI I~

Structurally MH closely resembles the pyrimidine base uracil. Cradwick

[12], on the basis of its crystal structure and possible formation of base pairs
with nucleic acid bases (both purines and pyrimidines), suggested that the
c o m p o u n d can be incorporated into R N A and may interfere with incorporation
of any nucleoside into RNA. The possibility of MH incorporation into DNA
was n o t excluded by the author.
The results of chemical analysis of MH and the methods applied for the
analysis of its residues have been discussed in detail by Haley [35]. Among
several methods developed for quantitative determination of MH residues in
tobacco smoke, food and crops, gas chromatography seems at present the most
sensitive [34].

Effect on cellular metabolism

Plants
MH is readily absorbed both b y roots and leaves, although the uptake differs
considerably between plant species [105]. According to Coupland and Peel [9,
11] and Smith et al. [105], the c o m p o u n d accumulates preferentially in areas
of active growth. The dividing cells are generally considered to be the place of
the primary action of MH in plants.
It has been established that, in several plant species, MH interferes with the
metabolism of nucleic acids and proteins. Povolotzkaya [86] observed inhibi-
tory effects of MH on the synthesis of nucleic acids in germinating p o t a t o
tubers, the effect being greater in isolated meristems than in intact tissues.
Evans and Scott [23] reported the inhibition of DNA synthesis b y MH in Vicia
18

faba: they used an autoradiographic m e t h o d with 3H-labelled thymidine. In the

study of cell-cycle duration these authors proved that MH treatment extends
the DNA synthesis period by a b o u t 13 h.
Lobov [58] found that, in pea seedling roots, a 6-h incubation with 10 -2 M
MH resulted in 50% inhibition of DNA and protein synthesis, while synthesis of
RNA was less affected. Nood~n [81] found that MH inhibits DNA and RNA
synthesis in corn seedling roots but has little or no effect on the uptake of
radioactive precursors [3H]thymidine and [3H]uridine. According to Nood~n
[81,82], MH in corn roots affects DNA synthesis sooner than that of RNA,
whereas protein synthesis is reduced only after a still longer exposure. This
author suggested that MH preferentially inhibits the synthesis of rRNA with
t R N A synthesis relatively unaffected. A significant decrease in nucleic acid and
protein content was reported by Rakitin and Strelnikova [90] and Rakitin et
al. [92] for germinating potato tubers derived from plants exposed to MH
before harvest. In buds of germinating tubers the contents of RNA, DNA and
protein were a b o u t 20, 3 and 10 times lower than in control buds.
It was postulated by Lobov [58] and by Lobov et al. [60] that, in plant
cells, MH can replace a pyrimidine or purine base in DNA precursors, both
nucleosides and nucleotides. If this is true, nucleotides in which the nucleic
acid base is replaced by MH can block the activity of DNA polymerase,
resulting in a decreased rate of DNA replication. Another possibility is that
inhibition of DNA synthesis is due to incorporation of MH-containing nucleo-
tides into the growing polynucleotide chain.
Although incorporation of MH into DNA is merely a hypothesis, its incorpo-
ration into R N A has been confirmed experimentally. Coupland and Peel [9]
reported incorporation of 14C-labelled MH into R N A in r o o t tip cells of willow
Salix viminalis. Selective accumulation of 14C-MH in plant nucleoli, as observed
on autoradiograms by Callaghan and Grun [7], also seems to be connected with
incorporation of MH into R N A (particularly into rRNA). On the other hand,
Lobov et al. [60] were unable, by spectrophotometric methods, to detect
incorporation of measurable amounts of MH into DNA or R N A of several plant
species.
MH is known to be an antagonist of uracil. Coupland and Peel [10] found
that, in willow, MH gives a b o u t 50% inhibition of uracil uptake. The type of
inhibition, similar to that of competitive inhibitors of enzymes, suggested that
MH suppresses the uptake of uracil into cells by a competitive process. On the
other hand, no inhibition of MH uptake by uracil was noted. Study with some
derivatives of MH showed that any modification of the MH structure resulted
in a certain loss of ability to concentrate in r o o t apices and to inhibit uracil
uptake [11]. Several data show that, in plants, inhibitory effects of MH can be
reversed by uracil [6,56,86], by thymine and a mixture of bases and partially
by adenine and guanine [69]. These findings strongly suggest that the inhibi-
tory effects of MH on plant growth result primarily from its interference with
nucleic acid metabolism.
This view is supported b y the cytological observation of Lobov et al. [59]
performed b y the m e t h o d of time-lapse microphotography. In tissue culture of
the apple tree Malus domestica grown in medium containing 10 -2 M MH, during
the first 2 h, characteristic changes in the behaviour of the nucleus were
 19

observed. After 4--6 h the nucleus decreased in size, while aggregation and dis-
integration of cytoplasm were noted n o t earlier than 8 h after the beginning of
exposure to MH.
Numerous studies have been recorded on the inhibitory effects of MH on
plant enzymes, e.g. lipase [4,75], starch phosphorylase and amylase [42],
peroxidase, phosphatase and polyphenolase [36], and on enzymes connected
with oxidative metabolism and respiration [89,91,107]. Interactions of MH
with auxins and other growth regulatory hormones have also been reported
[32,86]. A comprehensive review on the effects of MH on cellular processes
other than metabolism of nucleic acids and protein has been presented by
Hoffman and Parups [39]. Various aspects of the inhibitory effects of MH on
cellular metabolism and plant growth have been discussed b y Rakitin et al.
[891.
According to the available data, higher plants can metabolize MH to some
extent. Towers [113] found t w o u n k n o w n radioactive spots on chromatograms
and autoradiograms of extracts of wheat seedlings exposed to 14C-MH, one of
which was identified as the H-glycoside of MH. Biswas et al. [5] have iden-
tified in the tea plant Camellia sinensis 4 possible metabolic products of MH:
lactic acid, succinic acid, maleimide and hydrazine. Some enzymic reactions
(oxidation and/or reduction requiring oxidase, peroxidase and dehydrogenase)
were suggested to be responsible for cleaving the MH ring structure and its
detoxication.
Rakitin et al. [89] assumed that detoxication of MH in plants can be
achieved by biochemical degradation of its molecule, by binding of the com-
p o u n d with proteins and other metabolites as well as by adsorption of MH by
the cell constituents and partly b y release into the environment. Binding of 14C-
MH in basal parts of maize roots was also postulated by Nood~n [82] to be a
possible mechanism for detoxication of MH in plants.

Animals
Only few data are available on the effects of MH on the metabolism of ani-
mal cells. According to the early work of Barnes et al. [3], in rabbits exposed
to an oral dose of 100 mg/kg, 43--62% o f MH was excreted unchanged within
the first 48 h. After the dose of 2 g/kg the excreted MH was characterized as its
benzylamine salt, and no other metabolite was identified. The authors con-
cluded that 40% MH m a y either be excreted more slowly or else broken d o w n
and degraded. According to Mays et al. [40], after oral administration of 14C-
MH to rats, less than 0.001% of the radioactivity was found in blood and
tissues, 0.2% 14CO2 in expired air, 12% in faeces and 77% in urine. The urinary
excretion contained 92--94% MH and 6--8% of an u n k n o w n conjugate of MH.
These results suggest that the ability of mammals to metabolize MH is very
limited. Nelson and Kearney [80], studying the metabolism o f MH by hepatic
microsomes from phenobarbital-induced rats, came to the conclusion that the
mammalian liver microsomal system is incapable of metabolizing MH.
In a recent publication, Fritz and Lippert [26] reported that, in Yoshida-
Sarcoma Ascites cells, MH decreased the incorporation o f the labelled precur-
sors into DNA, R N A and proteins by a b o u t 30%, both in vivo and in vitro.
These findings suggested that MH can be c y t o t o x i c in animal cells w i t h o u t any
need to be activated in the organism.
20

Induction of chromosomal aberrations

Plan ts
Chromosome-breaking effects of MH in plants were first described by Dar-
lington and McLeish in 1951 [15]. According to their data and those reported
later by McLeish [72] in Vicia faba, MH-induced chromosomal aberrations were
preferentially localized in heterochromatic segments; the effect was of the
delayed type and the aberrations, only of chromatid type, were produced with
normal rejoining of broken ends. Chromosome-breaking effects of MH in plants
have been confirmed for a number of plants species such as oat, maize, soy-
bean [8], barley [45,64,87], Crepis capillaris [19,4], Ntgella domescena [78],
Allium cepa [73] and Alo~ vera [31].
Localization of MH-induced chromosome damage in heterochromatic regions
has been studied in detail by Michaelis and Rieger [74] and by Rieger [94]
with the use of the new k a ~ o t y p e of V. faba characterized by transfer of indi-
vidual chromosome segments to another position within the karyotype. The
transfer of chromosome segments in which aberrations were preferentially
localized was accompanied by a corresponding shift in aberration distribution.
Preferential localization of aberrations induced by MH in heterochromatic seg-
ments of Vicia chromosomes was also reported by Slotova et al. [104], Natara-
jan and AhnstrSm [78] and by Zuk and Swietlifiska [120]. Graf [28] also oh-
served in maize a distinct correlation between the number of anaphase bridges
and that of heterochromatic knobs in the variety studied.
Precise data concerning the chromosome-breaking effects of MH, depending
on the cell cycle, were obtained by Evans and Scott [23] in autoradiographic
experiments with Vicia faba. These authors found that cells exposed to MH
whilst in G2 stage were not delayed in their development to the first post-
treatment mitosis and contained no aberrations. However, when observed at
the second post-treatment mitosis, these cells contained aberrations of chro-
matid type. Cells exposed while in S stage were considerably retarded and con-
tained aberrations only of chromatid type. Chromatid-type aberrations in the
first post-treatment mitosis were also found in cells exposed to MH in G~ stage.
It was clear that structural changes in chromosomes were produced only
during the S stage and, according to the authors, "were consequences of errors
in chromosome duplication occurring during the DNA synthesis phase". These
results were confirmed by Scott [103] and by Nayar [79].
The frequency of chromosomal aberrations induced by MH is strongly
potentiated by caffeine and other methylated oxypurines. According to Kihl-
man et al. [51--53], Sturelid and Kihlman [106], Swietlifiska and Zuk [108]
and Osiecka and Swietlifiska [84], post-treatment with low concentrations of
caffeine dramatically increased the aberration yields induced by MH, parti-
cularly in cells exposed to caffeine while in S stage of the cell cycle. In such
cells the frequency of chromatid exchanges was enhanced more drastically than
the frequency of other types of aberrations [108]. Similar potentiating effects
of caffeine were described for aberrations produced by a number of mono-,
bi- and poly-functional alkylating agents [ 51,52,108].
The preferential localization of MH-induced structural changes in hetero-
chromatic segments of chromosomes, production of aberrations only during
 21

DNA replication as well as strong potentiation of aberration frequency by caf-

feine, closely resemble the chromosome-breaking action of bifunctional alky-
lating agents: nitrogen mustard (HN2) [24], d i e p o x y b u t a n e [109,119] and
mitomycin C, which is also k n o w n to act as an alkylating agent [93]. Produc-
tion of chromosomal aberrations by bifunctional alkylating agents has been
attributed to the properties of linking complementary strands and misreplica-
tion of DNA at corresponding sites. The action of MH on chromosomes, similar
to that described for alkylating agents, as well as its inhibitory effects on DNA
synthesis, strongly suggest a chemical interaction of the c o m p o u n d with DNA
during the replication period. It is, therefore, n o t surprising that caffeine,
known as an inhibitor of DNA repair, enhances chromosome-breaking effects
of MH.
The frequency of chromosomal aberrations induced by MH in plants
depends strongly on pH and temperature of treatment. According to Kihlman
[49], in Vicia the yield of aberrations is a b o u t 4 times higher at pH 4.7 than at
pH 7.3 and increases with temperature. Chromosome-breaking effects of MH
can be inhibited by respiratory inhibitors such as cyanide, carbon m o n o x i d e or
azide [50] and enhanced b y the presence of oxygen, although the c o m p o u n d
has also a considerable effect under anaerobic conditions. It was reported by
Loveless [61] that, in Vicia, the rate of production of chromosomal aberrations
by MH was n o t affected by pre-, post- or simultaneous treatments with 10
times the concentration of uracil, thymine or orotic acid. The observation of
Coupland and Peel [10] that uracil does n o t inhibit the uptake of MH seems to
explain the data of Loveless on the lack of effect of uracil on production of
chromosomal abnormalities by MH. According to Kaul and Choudhary [47],
the chromosome-breaking effects of MH were decreased in onion and barley by
sodium thiosulphate; a similar protecting effect of thiol groups was also
reported for alkylating agents. According to Valadaub-Barrieu and Izard [ 115],
in Vicia the frequency of MH-induced aberrations was decreased to some
extent by acrolein.

Animals
The first reports on induction of chromosome damage by MH in animals
appeared only as late as 1971. Manna and Das [65] observed gaps, constric-
tions, chromatid and chromosome breaks and translocations in bone-marrow
cells of mice injected with MH. These results were confirmed in a more recent
study by Manna et al. [66]. According to Manna and Parida [67], administra-
tion of MH to grasshoppers as a f o o d c o m p o n e n t resulted in the appearance of
chromosomal aberrations in spermatocytes. The 5 species under study differed
in sensitivity to the treatment, b u t there was no correlation between sensitivity
and chromosome length or number. In grasshoppers, in contrast with plants,
heterochromatin (X chromosome) was n o t selectively broken b y MH. Study of
chromosome-breaking effects of MH in the fish, Umbra limi, have shown that
long-term exposure to the chemical caused a slight b u t significant dose-depen-
dent increase in chromosome damage [55]. In some experiments, however, MH
was inactive in inducing c h r o m o s o m e damage in mammalian tissues. According
to Kihlman et al. [51], MH did n o t produce any chromosomal aberrations in
cell cultures of the Chinese hamster CL-I. Similarly, Perry and Evans [85] were
22

n o t able to induce either chromosomal aberrations or sister-chromatid

exchanges in cultures of CHO cells exposed to MH. Timson [112] observed a
reduction of the mitotic index in human l y m p h o c y t e s cultured in vitro and
exposed to MH, b u t the appearance of chromosomal aberrations was not
recorded. Also, Hitachi et al. [38] reported that MH did n o t induce chromoso-
mal aberrations in rat-liver cells.

Sensitivity to the toxic effects o f MH

Plan ts
Higher plants are particularly sensitive to MH, and respond to its action by
growth retardation, which, depending on the dose and length of exposure can
be only temporary or lethal. However, among higher plants there are clear
species-specific differences in sensitivity to the toxic effects of MH.
Selective action of MH on higher plants was first described by Currier and
Crafts [14] in 1950. These authors observed strong inhibition of barley growth,
whereas in the same conditions c o t t o n was apparently unaffected. It was also
found that y o u n g plants usually responded more intensively to MH than older
representatives of the same species. MH has been used as a non-selective grass
killer owing to its ability to kill y o u n g plants of every species [13,37]. Some
data indicate, however, different species-specific sensitivity to MH among both
mono- and di-cotyledons. This clearly appears in the 15-year study of Willis
[117] on ecological changes in road-side vegetation exposed to MH. Selective
sensitivity to this herbicide resulted in long-term changes in species composi-
tion on the experimental plots.
Among lower plants, inhibitory effects of MH have been described in ferns
and algae. According to Khave and R o y [48], MH depressed spore germination,
cell division and growth of the fern Dryopteris cochleata. Sarma and Shyam
[100] reported inhibitory effects of MH on cell division and growth in the alga
Gymnodinium inversum (Dinophyceae). Similar results were obtained by
Dodge [17] for Prorocentrum micans (Dinoflagellatae). Among Charophyta,
MH was found to be c y t o t o x i c to some species belonging to Chara and Nitella.
Cytological effects (pycnotic nuclei) were produced, however, only by rela-
tively high concentrations of MH, suggesting that algae are more resistant to
this chemical than are higher plants [101].
It can be assumed that differential sensitivity to MH observed among plant
species depends both on their ability to take up and degrade MH by plant
metabolism as well as on the efficiency of some detoxicating mechanisms as
discussed earlier.

Animals
Among animals, mammals are highly resistant to the toxic effects of MH and
can tolerate high doses of the drug [3]. Haley [35], in his review on maleic
hydrazide, quotes data from the unpublished reports of Tare [110,111],
showing very low toxicity of the chemical to rats, dogs and cows under chronic
and subchronic ingestion. According to Tate's reports, the diethanolamine salt
of MH was more toxic to rats than the sodium salt (LDs0 values 2.35 and 6.95
g/kg respectively).
 23

According to Luckens and Wattimena [62], the diethanolamine salt of MH,

applied as a pretreatment, markedly increased the toxicity of the insecticide
dieldrin. When this insecticide was used for pretreatment, the toxicity of
diethanolamine salt was markedly potentiated in female, but not in male, rats.
The authors suggested that the diethanolamine salt of MH might be metabol-
ized in the liver or be itself an enzyme inhibitor. Fischnich et al. [25] observed
a decrease in the level of rat fertility when the animals were kept on a diet
including tubers from p o t a t o plants sprayed with MH before harvest. No such
effects were noted when rats were fed with tubers exposed to MH after lifting.
This finding suggested that some metabolites of MH produced in plant tissues
were more toxic to animals than MH itself. No effect of MH on rat-liver cells
was reported by Mannel and Grice [68] and Hitachi et al. [38] in animals kept
on a diet with this chemical. McCarthy and Epstein [71], however, observed
inhibition of mitotic activity and unbalanced growth in a cell line established
from the mouse t h y m u s when exposed to MH at the concentration 1 × 10 -2 M.
Information on the toxicity of MH to representatives of other groups of
animals are incomplete. Kanehisa [46] observed a marked retardation of larval
development in Drosophila melanogaster exposed to MH. According to Manna
and Parida [67], administration of 5 mg MH per individual with the food
resulted in 20--60% mortality of grasshoppers, depending on the species tested.
The observation of Mjuge and Viglierchio [75], that MH, when added to an
extract of agarized soil, decreased the infestation of carrot seedlings with
Nematoda Prathylenus vulnus, suggests that nematodes can be controlled by
this chemical. Robinson [95] reported a decreasing effect of MH on fertility of
the pea aphid Acyrthosiphon pisum, particularly when the drug had undergone
metabolism in the plant. Toxicity and growth retardation in certain amphibians
such as salamander (Ablystoma punctata) and frog (Rana pipiens) exposed to
MH was reported by Greulach et al. [29]. In the same experiments, toxicity of
MH to some Crustacea, e.g. Cyclops and Daphnia, was noted. According to the
data presented it can be accepted that MH itself, or some of its metabolites in
plants, can be toxic to animals to some extent.

Microorganisms
The data on toxicity of MH to bacteria and fungi mainly concern soil micro-
organisms [39]. It is generally accepted that MH has only a slight effect on
microorganisms, enhancing or inhibiting growth, depending on the species
tested.
According to H o f f m a n et al. [41] and H o f f m a n and Parups [40], micro-
organisms in soil usually cause a rapid breakdown of MH. This was concluded
from the finding that the MH c o n t e n t in the soil decreases with time. Sarda-
rayan et al. [99] reported that MH applied in sterile soil was 3 times more effec-
tive in inhibiting growth of wheat seedlings than when applied in non-sterile
conditions. In the same experiments the growth of some bacteria and fungi was
enhanced by UV. The authors concluded that soil microorganisms are able to
decompose or degrade a significant proportion of MH, and the products can be
utilized as a source of nutrition. Degradation of MH by the soil microflora was
confirmed by the presence of newly formed but unidentified substances
revealed by chromatographic analysis of the culture media [98].
24

In experiments performed in our laboratory (Zuk and Swietlihska, unpub-

lished results), treatment with 0.2% MH during 60 min at pH 5.3 did not
reduce survival of the diploid strain of yeast Saccharomyces cerevisiae and did
n o t change the frequency of conversion in the threonine locus in this strain.
Neither was the survival of Escherichia coli reduced in full growth medium
supplemented with 0.25% MH.

Mutagenesis

MH has n o t been studied extensively for its ability to induce point mutation
either in prokaryotes or eukaryotes. According to the few data available, it
appears to be mutagenic in some systems, but in others no point mutations
were induced.
Northrop [83] found MH to be mutagenic in the bacterium Bacillus me-
gatherium 20A, increasing two-fold the proportion of virus-producing cells as
well as streptomycin-resistant cells. In another microbial system, where T4 bac-
teriophage was used to detect MH-induced mutations of rII type, the frequency
of point mutations was n o t increased over the spontaneous m u t a t i o n rate,
although two known mutagens, 5-bromouracil and 2-aminopurine, were highly
mutagenic in this system [2].
According to Nasrat [77], in Drosophila the frequency of sex-linked reces-
sive lethals in the progeny of males reared on food containing 0.4% MH was
slightly increased as compared with the control (1.07% and 0.5%, respectively).
Epstein et al. [21], in their study on the mutagenicity of a number of chemicals
by the dominant-lethal assay in the mouse, classified MH as an agent " n o t
meeting any screening criteria for mutagenic effects".
In higher plants, MH produced a low frequency of chlorophyll mutations in
barley, and accordingly was claimed to be a weak mutagen as compared with
N-nitroso-N-methylurea [63]. On the other hand, Sanz and Hacke [97] did n o t
observe induction by MH of resistance to wheat rust Puccinia graminis in wheat
plants.

Carcinogenesis

The potent chromosome-breaking action of MH observed in plant cells as

well as the widespread use of this chemical in agriculture has raised the problem
of its possible carcinogenicity to animals and also to man. Early work on the
carcinogenic properties of MH in laboratory animals, mice and rats, gave nega-
tive results [3,96]. These findings, however, have n o t been confirmed in recent
publications and the carcinogenicity of MH was even claimed to be " a t variance
with the data on which it was based" [20].
In 1968 Epstein and Mantel [22] published data which clearly indicated the
high carcinogenicity of MH for male mice. In animals injected subcutaneously
with aqueous solutions or tricaprylic suspension of MH as free acid, hepatomas
developed in 18 of 26 infant male mice, in contrast with 4 of 48 control mice.
According to Dickens and Jones [16], MH is also carcinogenic to rats, as 3 out
of 6 animals developed sarcomas after twice-weekly injections of MH in arachis
oil for up to 65 weeks.
 25

On the other hand, Akin [1] f o u n d MH was not tumorigenic to mouse skin
and decreased by about 50% the tumour-initiating action of the carcinogenic
hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA). In Akin's experi-
ments, MH was applied to mouse skin as a water solution, and it is possible that
in such conditions its action differed from that exerted after subcutaneous
injection. In IARC Monographs on the evaluation of carcinogenic risks to man
[46], MH was also reported n o t to be carcinogenic to one or more laboratory
rodents. In Drosophila melanogaster, Kanehisa [46] did n o t observe any effects
of MH on the incidence of tumours.
Some data on toxicity of MH to certain animals seem, however, to be impor-
t a n t in considerations of possible carcinogenic properties of the c o m p o u n d .
The suggestion that some metabolites in plants exposed to MH were more toxic
to rats [25] and pea aphids [95] than MH itself points to the need for evalua-
tion of plant extracts or homogenates for possible carcinogenic properties in
animals. A well-known carcinogen and mutagen, hydrazine [54], identified as a
possible metabolite of MH in tea plants [5], might be responsible for the
presumable mutagenic and carcinogenic action of plant extracts.

Uses in agriculture and the food industry, and the genetic risk to man

MH was introduced into agriculture and the food industry as a p o t e n t plant

growth regulator since 1950 [14,116,118]. The main usage of MH is that for
inhibiting sprouting of vegetables and stored root crops, inducing male sterility,
delaying of flowering, prolonging of the period of d o r m a n c y and increasing of
frost resistance in crops and trees. Owing to selective toxicity of MH to plant
species and the high sensitivity of y o u n g plants of any species to this com-
pound, MH has become a major commercial herbicide. The widespread and
numerous applications of MH have been reviewed by Crafts [13] whose review
has been supplemented by Hoffman and Parups [39].
In tobacco farming, MH is used t h r o u g h o u t the world as an inhibitor of
suckers on tobacco plants. It has been estimated that as much as 80% of Ame-
rican-grown tobacco is treated with this chemical [114]. A combined applica-
tion of MH and nitrogen fertilizers was reported to increase the yield of
tobacco leaves by up to 25% [30]. MH applied to potato plants before harvest
is known to inhibit sprouting and increase the quality of tubers that are to be
used as food [76]. Prokudina [88] reported a significant increase in the yield
of melons due to the effective control of broom rape Orobanche by MH. The
contents of glucose, fructose and other sugars in fruit were also increased.
Dolnicki [18] found t h a t t r e a t m e n t of seeds of winter wheat with MH
increased the frost resistance of the plants by about 10%. Similarly, suppression
of a u t u m n growth in lemon trees by spraying with MH was reported to
decrease considerably the winter damage to the shoots [27]. The applications
of MH listed above, all recently published, point to the widespread and progres-
sive usage of the c o m p o u n d as a means of increasing plant productivity.
In view of the foregoing considerations, it can be accepted that the entire
h u m a n population of civilized countries is continuously exposed to MH from
both food and cigarettes. MH residues in tobacco and other crops have been
estimated as relatively high [75]. Over 1/~g of the 30 pg of MH in an average
26

American cigarette has been shown to be transferred unchanged to the main-

stream smoke [57] and a b o u t 99% of the MH in cigarette tobacco to be
degraded to other products [34]. These data raise the problem of the possible
contribution of MH to the carcinogenicity of cigarettes. Epstein and Mantel
[22] estimated the total annual MH dose to which humans are exposed as
a b o u t 630 mg which is some 12 times more than the a m o u n t found to be car-
cinogenic to mice after parenteral administration. It seems, however, that inges-
tion of MH via the oral route decreases considerably the hazardous possibilities
of MH carcinogenicity to man. This may be because, in mammals, a predomi-
nant proportion of MH is excreted unchanged within a short time [3,70].
The need for further tests, including biotransformation studies and pharma-
cokinetic studies, to ascertain the possible carcinogenicity of MH to humans is
strongly emphasized by Haley [35].

Acknowledgements

Thanks are due to Mr. John Wasson, Environmental Mutagen Information

Center, Oak Ridge, TN, for supplying a print-out of the relevant literature and
also numerous reprints of publications.

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28

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