J. Cell Sci. io, 15-25 (1972) 15

Printed in Great Britain

REGULATION OF MITOSIS

m . CYTOLOGICAL EFFECTS OF 2,4,5-TRICHLORO-

PHENOXYACETIC ACID AND OF DIOXTN CONTAMINANTS

IN 2,4,5-T FORMULATIONS

W. T.JACKSONDepartment of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755,

U.S.A.

SUMMARY

The conclusion of earlier studies that the pronounced cytological effects of 2,4,5-trichloro-phenoxyacetic acid (2,4,5-T) formulations are actually due to 2,4,5-T is unwarranted. Inhibitionof mitosis and the development of cytological abnormalities are caused by a contaminant,2»3>7>8-tetrachlorodibenzo-/>-dioxin. The appearance and behaviour of dividing endospermcells of the African blood lily (Haemanthiu katherinae Baker) subjected to a io"4 M solution ofhighly purified 2,4,5-T, observed in vitro by phase-contrast microscopy for a z-h treatmentperiod, did not differ significantly from controls. Subjecting these cells to 0 2 or i-o fig/\.dioxin, to 0 2 /tg/1. dioxin plus io"4 M 2,4,5-T (highly purified sample), or to io^1 M 2,4,5-Tcontaining dioxin as a contaminant caused a dramatic inhibition of mitosis. These preparationsalso induced formation of dicentric bridges, chromatin fusion with formation of multinuclei ora single large nucleus. Impairment of the functioning of the mitotic apparatus was evident;chromosomes ' floated free' in the cytoplasm. Both quantitative and qualitative data sub-stantiate these conclusions.

In view of the extreme toxicity of dioxins to man, of their demonstrated teratogenic effects insome animals under experimental conditions, of their effectiveness in producing cytologicaleffects in dividing cells formerly ascribed to 2,4,5-T, of their presence as contaminants in2,4,5-T, pentachlorophenol, and other polychlorophenols, and of the potential for widespreadcontamination of the environment with dioxins, investigation of all facets of this class of com-pounds should be given high priority. It is quite possible that contamination of the environ-ment with dioxins from sources of polychlorophenols is far greater than that contributed by2,4,5-T. Amplification of dioxin levels in food chains is, of course, of particular concern.

INTRODUCTION

In the intervening years since Hamner & Tukey (1944) first recognized the selectiveherbicidal properties of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), literally hundredsof investigations have been conducted to exploit its potential as a herbicide, to deter-mine its biochemical effects, to investigate its metabolic fate, to unravel the pathwayof its degradation in soil, and to elucidate its mechanism of action at the cytologicallevel (see, for example, Woodford, Holly & McCready, 1958; Hilton, Jansen & Hull,1963; Andreae, 1963; Audus, 1964; King, 1966; Hull, 1967; Fryer & Evans, 1968;Kearney & Kaufman, 1969; Casida & Lykken, 1969). Although it has been recognizedfor over 20 years that preparations of 2,4,5-T contain contaminants (Anon, 1970), ithas been generally assumed that the herbicidal effects are due to the 2,4,5-T; and not

16 W.T. Jackson

to any contaminant present. The purpose of the present study was to determinewhether highly purified 2,4,5-T produced the cytological effects in dividing cells pre-viously attributed to 2,4,5-T.

MATERIALS AND METHODS

A detailed description of methods and materials, characterization of the stages or events ofmitosis, and a quantitative and qualitative evaluation of the behaviour of cells under controlledconditions were presented earlier (Jackson, 1967). Some modifications in the original methodswere made in a study of the interaction of isopropyl N-phenylcarbamate and melatonin (Jackson,1969). Techniques for studying the ultrastructure of dividing endosperm cells have also beendescribed (Hepler & Jackson, 1968, 1969). Young seeds in the developing fruits of the Africanblood lily (Haemanthus katherinae Baker) possess liquid endosperm. When the seeds haveattained a length of 6-8 mm the endosperm contains hundreds of uninucleate cells lacking cellwalls. If this liquid is spread on a 0-5 % agar surface containing 3 5 % glucose, and the excessliquid allowed to drain, the cells will adhere to the agar surface and become very flat. Mitosisin these flattened cells can be observed easily by phase-contrast microscopy. The effect of achemical, or chemicals, is studied by incorporation into the agar solution before making a pre-paration, or by perfusing the solution across the cells in a chamber while they are under obser-vation. Mol6-Bajer (1955) has calculated that dilution of the chemical in agar by the liquid ofthe endosperm would reduce the effective concentration to approximately one-half of theoriginal. Dilution in the perfusion chamber should be of minor importance. The 2-h obser-vation period in the present study was selected to ensure detection of a possible acceleration ofmitosis in cells subjected to treatment. For each treatment, data are presented as the per-centage of cells selected in a given mitotic stage that proceeded to the indicated subsequentstage during the 2-h treatment period.

Because it could not be demonstrated that the assumptions underlying the use of parametricstatistical procedures could be met (including (1) that variance homogenity exists, (2) that thedata follow a normal distribution, even though they were selected randomly, and (3) thatmeans and variances are not associated), the non-parametric Mann-Whitney U test (see Siegel,1956) was applied to the data to determine if significant differences existed between treatments.The null hypothesis (Ho) in each test performed between 2 sets of treatment data was that therewas no significant difference between the 2 treatments with respect to the percentage of cellsprogressing from an initial mitotic stage to a subsequent one. The alternative hypothesis (Ht)was that the 2 sets of data being compared were significantly different from one another. Direc-tion of significant differences was not taken into account in this application of the Mann-Whitney U test; thus, the test was applied as a 2-tailed test. A 5 % level of significance wasestablished as the basis of accepting or rejecting the null hypothesis. When 2 sets of data werefound to be different from one another at the a = 5 % level, the phrase ' significantly different'has been used in the text; at the a. = 2 % level, or less, the phrase ' highly significantly different'has been applied. Tests performed which did not indicate significant differences at levels of5 % or more were assumed to reveal differences due to random error or chance variation.

It should be emphasized that transforming the actual number of cells reaching a specificmitotic stage to a percentage, based on the total number of cells observed initially at an earlierstage, introduces an unusual statistical bias for the data couplets being compared. In data sets(1) in which single cells do progress from one mitotic stage to a subsequent one, or where singlecells do not proceed to the next stage, or (2) where all of a large number of cells in a singlechamber do or do not reach the next mitotic stage, all cell numbers, regardless of sample size,are transformed to a percentage of o or 100 %. Since the Mann-Whitney U test is based on aprocedure of ranking data, ranks of o and 100% represent extremes and are frequently respon-sible for ties occurring in the ranks. Ties of this nature, particularly for single cell observations,tend to influence the test by allowing one to accept the null hypothesis when the empirical data,in fact, may suggest that significant differences do exist. Thus, in many instances the levels ofprobability (P) are actually lower than the values derived and presented in the text, due to thebias.

2,4,5-^ and dioxin effects on dividing cells 17

To decrease the possibility of error in actual computation of statistics, the GE-635 computerat the Dartmouth Kiewit Computation Center was used for analysis.

RESULTS AND DISCUSSION

The cytological effects attributed to 2,4,5-T are quite dramatic. Croker (1953)reported that the effects of 2,4,5-T on mitosis in Allium cepa are proportional to con-centration and can be grouped as physiological and structural effects. She concludedthat 2,4,5-T caused stickiness and condensation of chromosomes and delay in spindleformation within 2 h after treatment. Bradley & Crane (1955) reported that treatmentof apricot trees with 2,4,5-T as a spray at the beginning of pit-hardening in the fruitscaused cell enlargement and a large increase in number of cells of the high polyploidclass over the controls. Hull (1957) concluded that 2,4,5-T induced incipient cellproliferations in phloem parenchyma cells within 48 h after treatment, but only withthe lowest concentrations of 2,4,5-T in combination with a relatively low concentrationof wetting agent. More recently, Sawamura (1964) reported that treatment of Trades-cantia hair cells with 0-05 % solution of 2,4,5-T amine salt caused formation of multi-nucleate cells with multisepta.

Results of the present study (Table 1) reveal that the dioxin contaminant, not2,4,5-T, is responsible for most of the inhibition of the normal progression of mitosisand for the appearance of abnormalities in dividing endosperm cells of Haemanthuskatherinae Baker. The significance, or lack of significance, of difference in ability ofcells subjected to 2 different treatments to proceed from one phase of mitosis to anotheris given in Table 2. For example, 87% of the control cells proceeded from prophase(P) to prometaphase (PM) during the 2-h treatment period, whereas 80 % of thosesubjected to Dow's Analytical Grade (DA) I O ^ M 2,4,5-T and only 16% of thosesubjected to Amchen's 98% Purity (Am.) I O ^ M 2,4,5-T made this progression(Table 1). Those cells subjected to DA were significantly inhibited, whereas thosesubjected to the Am. material were highly significantly inhibited compared with thecontrols (Table 2). DA, compared with controls, significantly inhibited progressionfrom P to PM (80 v. 87%), from PM to cell plate formation (CP) (15 v. 48%) andnuclear membrane formation (NM) (8^.42%), and from metaphase (M) to NM(52 v. 77%)- There was no significant difference between controls and DA in 8 out ofthe 12 comparisons possible. The DA sample has less than o-2/ig/g of 2,3,7,8-tetra-chlorodibenzo-p-dioxin as a contaminant. Since the molecular weight of 2,4,5-T is255-5, a contamination of 0-2 /tg/g dioxin in the 2,4,5-T used to make a io~* M solutionwould result in a solution containing 0-005 /*§/!• dioxin. Although one cannot provethat the dioxin contaminant in DA is responsible for inhibition of the 4 progressionsmentioned above, one can state that if the level of dioxin is 0-005 /*g/l- o r lower, thenio~^ M 2,4,5-T is without effect in 8 of the 12 progressions studied. Furthermore,it should be pointed out that the 4 stages that were inhibited involved membranedisorganization or resynthesis; not chromosome movement. That is, io^1 M 2,4,5-Tdoes not interfere with chromosome movement in prometaphase or anaphase, orwith the separation of kinetochores at onset of anaphase. Nor does this rather high

20 W. T. Jackson

concentration of 2,4,5-T interfere with cell plate formation of cells that were in meta-phase at initiation of treatment, or with either CP or NM of cells already in anaphasewhen treated.

The maximum solubility of 2,3,7,8-tetrachlorodibenzo-p-dioxin in water is about0-2 /tg/1., but it is known that it is adsorbed to glass surfaces and, of course, in oursystem it would be diluted to approximately one-half of its original concentration bythe addition of the endosperm liquid. Thus the cells subjected to 'o-2/tg/l. dioxin'(Tables 1, 2) were actually subjected to even lower concentrations, perhaps less thano-i /tg/1. Cells subjected to 'i-o/tg/1. dioxin' were probably bathed in an approxi-mately water-saturated solution (i.e. 0-2 /tg/1.) of dioxin. Both solutions stronglyinhibited most stages of mitosis in the endosperm cells. In fact, inhibition by i-o /tg/1.dioxin was highly significant compared to controls in 11 of the 12 stages and significantin the other (Table 2). The solution containing 0-2 /tg/1. dioxin caused highly signi-ficant inhibition in 7 of the 12 stages, significant inhibition in 2 more, and did notsignificantly inhibit the remaining 3 stages. Inhibition by a solution containing 0-2/tg/1. dioxin plus DA io"4 M 2,4,5-T (undoubtedly containing some dioxin as a con-taminant) was of the same order of severity, compared to the controls, as i-o/tg/1.dioxin (Tables 1, 2). As pointed out previously, the effect of the io~* M 2,4,5-T (DA)in this treatment could be due to the 2,4,5-T itself, to the dioxin contaminant, or to acombination of the two.

Although the level of dioxin contamination in the Dow Chemical Company'sanalytical sample of 2,4,5-T was below the level of detection (0-2 /̂ g/g) the commercialgrade sample (lot 120499) from this company has approximately 0-5 /tg/g of 2,3,7,8-tetrachlorodibenzo-p-dioxin. The sample of 2,4,5-T (Amchem) has a purity of 98%or better. It should be emphasized that the Amchen sample is at least 3 years old andmay not be representative of the purity of their current production, if any. Examina-tion of Tables 1 and 2 reveals that both the Dow Commercial 2,4,5-T and the Amchemsample cause greater inhibition of mitosis than the Dow Analytical Grade of 2,4,5-T.There was no significant difference between the inhibitory effects of Dow Commercialand the Amchem sample when their effects on the 12 progressions in mitosis werecompared.

As might be expected, dioxin also induces cytological abnormalities that wereascribed to 2,4,5-T in earlier studies. Fig. 1 is a general view of dividing endospermcells as they appear in phase contrast when dividing on the control medium of 3-5 %glucose plus 0-5% agar. A sequence of photographs of a single cell progressingthrough mitosis on the control medium, with an analysis of time spent in each stage, isgiven in Jackson (1967). Fig. 2 in the present study is a general view of dividing cells208 min after they were subjected to io"4 M 2,4,5-T (DA). They were in prophase atinitiation of treatment and apparently dividing normally. Fig. 3 is of the same cells1 h later; 5 cells appear normal, whereas 1 cell in anaphase seems to have 1 or morelagging chromosomes. Figs. 4-6 reveal the progression of a cell treated with io"4 M2,4,5-T (DA) 30, 86, and 145 min, respectively, after initiation of treatment; althoughthe cell has continued to flatten, and consequently has become larger in diameter, thereis no evidence of cytological abnormalities. The Amchem sample of 2,4,5-T at io"4 M

2,4,5-^ and dioxin effects on dividing cells 21

did not inhibit normal contraction of chromosomes during prophase and prometa-phase, but did prevent the alignment of chromosomes (Figs. 7, 8). Fig. 7 shows theappearance of the cell only 28 min after initiation of treatment; Fig. 8 reveals theappearance 65 min later. Obviously, such cells did not complete mitosis, and the usualpattern was fusion of chromosomes into a single mass. One of the most characteristiceffects induced by dioxin alone, or in combination with 2,4,5-T, was the formation ofdicentric bridges with subsequent fusion of daughter chromatids. This is illustratedby Figs. 10 and 11, in which the cell was subjected to 0-2 /*g/l. dioxin. The cell was inearly anaphase and normal in appearance when first observed 20 min after initiationof treatment; 64 min later (Fig. 10) many dicentric bridges had formed and the cellplate had made its appearance; and 1 h later (Fig. 11) the chromosomes had fused intoa single mass, with a dumb-bell appearance, constrained by a well developed cell-plate.

As might be expected, dioxin in the presence of io^M 2,4,5-T (DA) also inhibitsmitosis and causes cytological abnormalities (Figs. 9, 12). The cell shown in Fig. 9was normal in appearance and in early anaphase when first observed 25 min afterinitiation of treatment with o-2 /tg/1. dioxin and io~* M 2,4,5-T (DA). An hour later,it possessed many dicentric bridges; 2-5 h after initiation of treatment the chromosomeshad fused into a single mass (Fig. 9).

Many cells treated with dioxin alone, or in combination with \o~i M 2,4,5-T (DA),exhibited excessive vacuolation and protrusion of the cytoplasm into near-sphericaloutgrowths as exemplified in Fig. 12. When first observed 20 min after initiation oftreatment with o-2/ig/l. dioxin and I O ^ M 2,4,5-T (DA), this cell was normal inappearance and was in early anaphase; 1 h later it appeared as shown in Fig. 12.

Synthesis of 2,4,5-T was originally accomplished by heating equimolar quantitiesof 2,4,5-trichlorophenol and monochloroacetic acid with a slight excess of sodiumhydroxide and water until the solution was evaporated almost to dryness (Pokorny,1941). In the synthesis of the related herbicide, 2,4-dichlorophenoxyacetic acid, 2,4-dichlorophenol is substituted for 2,4,5-trichlorophenol (Pokorny, 1941). Since thedioxin is produced as a contaminant in the formation of polychlorophenols such as2,4,5-trichlorophenol, but presumably not in formation of 2,4-dichlorophenol, it isassumed that 2,4-dichlorophenoxyacetic acid is free of the dioxin contaminant. How-ever, until it is demonstrated that highly purified samples of 2,4-D are capable ofexerting effects attributed to 2,4-D samples available in the past, this compound, too,will remain suspect.

The valuable technical assistance of R. H. Harlow, T. L. Jackson and Mrs Maxine Bean isgreatly appreciated. I wish to acknowledge the aid of Dr Harold L. Allen and Dr T. E. Kurtzin devising a suitable statistical analysis of the data. Samples of 2,3,7,8-tetrachlorodibenzo-^)-dioxin and formulations of 2,4,5,-trichJorophenoxyacetic acid were provided by the DowChemical Company, the Amchem Chemical Company, and Dr Jack Plimmer of the UnitedStates Department of Agriculture. This research was supported by NIH grant 1R01ES00468-01.

22 W. T. Jackson

REFERENCES

ANDREAE, W. A. (1963). Metabolic Inliibitors, vol. 2 (ed. R. M. Hochster & J. S. Quastel),pp. 243-261. New York: Academic Press.

ANON (1970). Another herbicide on the blacklist. Nature, Lond. 226, 309-311.AUDUS, L. J. (ed.) (1964). The Physiology and Biochemistry of Herbicides. New York: Academic

Press.BRADLEY, M. V. & CRANE, J. C. (1955). The effects of 2,4,5-trichlorophenoxyacetic acid on cell

and nuclear size and endopolyploidy in parenchyma of apricot fruits. Am. J. Bot. 42,273-281.

CASIDA, J. E. & LYKKEN, L. (1969). Metabolism of organic pesticide chemicals in higher plants.A. Rev. PL Physiol. 20, 607-636.

CROKER, B. H. (1953). Effects of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxy-acetic acid on mitosis in Allium cepa. Bot. Gaz. 114, 274-283.

FRYER, J. D. & EVANS, S. A. (eds.) (1968). Weed Control Handbook, vol. 1 and 2. Oxford:Blackwell.

HAMNER, C. L. & TUKEY, H. B. (1944). The herbicidal action of 2,4-dichlorophenoxyaceticacid and 2,4,5-trichlorophenoxyacetic acid on bindweed. Science, N.Y. ioo, 154-155.

HEPLER, P. K. & JACKSON, W. T. (1968). Microtubules and early stages of cell-plate formationin the endosperm of Haemanthus katherinae Baker. J. Cell Biol. 38, 437—446.

HFPT.FR, P. K. & JACKSON, W. T. (1969). Isopropyl A^-phenylcarbamate affects spindle micro-tubule orientation in dividing endosperm cells of Haemantluis katherinae Baker. J. Cell Sci.5, 727-743-

HILTON, J. L., JANSEN, L. L. & HULL, H. M. (1963). Mechanisms of herbicide action. A. Rev.PL Physiol. 14, 353-384-

HULL, H. M. (1957). Anatomical studies demonstrating phloem inactivation and its dependencyupon the interaction of concentrations of 2,4,5-trichlorophenoxyacetic acid and an anionicwetting agent. PL Physiol., Lancaster, 32, Supplement 43.

HULL, H. M. (ed.) (1967). Herbicide Handbook of tlie Weed Society of America. Geneva andNew York: Humphries Press.

JACKSON, W. T. (1967). Regulation of mitosis in living cells. I. Mitosis under controlled con-ditions. Physiol. PL 20, 20-29.

JACKSON, W. T. (1969). Regulation of mitosis. II. Interaction of isopropyl iV-phenylcarbamateand melatonin. J. Cell Sci. 5, 745-755.

KEARNEY, P. C. & KAUFMAN, D. D. (ed.) (1969). Degradation of Herbicides. New York: Dekker.KING, L. J. (1966). Weeds of the World. London: Leonard Hill.MOLE-BAJER, J. (1955). A simple method for examining the action of chemicals on mitosis in

living endosperm. Acta Soc. Bot. Pol. 24, 619-625.POKORNY, R. (1941). Some chlorophenoxyacetic acids. J. Am. Chem. Soc. 66, 1768.SAWAMURA, S. (1964). Cytological studies on the effect of herbicides on plant cells. I. Hormonic

herbicides. Cytologia 29, 86-102.SIEGEL, S. (1956). Nonparametric Statistics for the Behavioural Sciences. New York: McGraw-

Hill.WOODFORD, E. K., HOLLY, K. & MCCREADY, C. C. (1958). Herbicides. A. Rev. PI. Physiol. 9,

311-358.

(Received 2 June 1971)

2,4,5-T and dioxin effects on dividing cells 23

For legend see next page.

24 W. T. Jackson

Figs. 1-12. Living endosperm cells of the African blood lily (Haemanthus katherinaeBaker) undergoing mitosis as observed by phase-contrast microscopy. Although thecells are spherical when suspended in the endosperm liquid in the seed, they flattenon the O'S % agar surface containing 3'5% glucose plus 2,4,5-T or dioxin as indicatedin the individual figure descriptions. A typical cell is approximately 6 /tm thick and150 /tm in diameter when fully flattened for observation. Chromosomes at meta-phase are about 3 fim in diameter.

Fig. 1. Cells now in various stages of mitosis were subjected to the control mediumcontaining 3 5 % glucose in 0 5 % agar for 165 min. Mitosis proceeded in a normalmanner in all cells, x 125.

Fig. 2. These cells were in early prophase at initiation of treatment. They weresubjected to the control medium containing io"4 M 2,4,5-T (Dow's Analytical Gradecontaining less than 0-2 /*g/g dioxin as a contaminant) for 208 min before this photo-graph was taken. Cells are now in metaphase and anaphase. x 125.

Fig. 3. Same field of view as in Fig. 2, photographed 269 min after initiation oftreatment, x 125.

Fig. 4. Cell in metaphase 30 min after initiation of treatment with Dow's analyticalgrade of 2,4,5-T at io"4 Min 3-5% glucose plus 0-5 % agar. x 355.

Fig. 5. Same cell as in Fig. 4 photographed 86 min after initiation of treatment.Note that the cell has continued to flatten and consequently has increased in diameter.x 355-

Fig. 6. Same cell as in Fig. 4 photographed 145 min after initiation of treatment.The cell has continued to flatten, x 355.

Fig. 7. Cell in late prometaphase 28 min after initiation of treatment with 10—' M2,4,5-T (Amchem sample) in 3-5 % glucose plus 0-5 % agar. Note that 2 chromosomesare already extending into the cytoplasm, x 465.

Fig. 8. Same cell as in Fig. 7 photographed 93 min after initiation of treatment.Note that several chromosomes are now floating free in the cytoplasm. Although con-traction of chromosomes has continued, there is no evidence that the spindle isfunctional. X465.

Fig. 9. This cell was normal in appearance and in early anaphase when first observed25 min after initiation of treatment with 0 2 /tg/1. dioxin and I O ^ M 2,4,5-T (Dow'sAnalytical Grade) in 3 5 % glucose plus 0-5% agar. When photographed (notshown) 67 min later it contained many dicentric bridges. The figure here reveals thatthe chromosomes have fused into a single mass by 152 min after initiation of treat-ment, x 500.

Fig. 10. This cell was normal in appearance and in early anaphase when firstobserved 20 min after initiation of treatment with 0-2 /tg/1. dioxin in 35 % glucoseplus 0 5 % agar. Note dicentric bridges and development of cell plate by 84 min afterinitiation of treatment, x 500.

Fig. 11. Same cell as in Fig. 10 photographed 144 min after initiation of treat-ment. Note development of cell plate, fusion of chromosomes, and decrease in dis-tance between poles, x 500.

Fig. 12. This cell was normal in appearance and in early anaphase when firstobserved 20 min after initiation of treatment with 0-2 /tg/1. dioxin and io"4 M 2,4,5-T(Dow's Analytical Grade) in 3-5% glucose plus 0-5% agar. Note dicentric bridgesand extreme vacuolation of cytoplasm by 85 min after initiation of treatment, x 705.

2,4,5-^ ond dioxin effects on dividing cells