changes in erythron organization during prolonged cadmium exposure: an indicator of heavy metal...

Changes in Erythron Organization during Prolonged Cadmium Exposure: An Indicator of Heavy Meta

Arthur Houston, Shelley Blahut, Ajrnal Murad, and Preetha Amirtharaj Department of Biological Sciences, Brock University, St. Catharines, One. L2S 3A 1, Canada

Houston, A., S. Blahut, A. Murad, and P. Amirtharaj. 1993. Changes in erythron organization during prolonged cadmium exposure: an indicator sf heavy metal stress? Can. I. Fish. Aquat. Sci. 58: 21 7-222.

Changes in erythron organization were examined in goldfish (Carassius auratus) exposed for 3 wk to 5, 1 5, and 25% sf 240-h Cd LC50. In a subsequent, more extended study, specimens were first rendered acutely anemic by immersion in phenylhydrazine HCI, allowed ta recover in Cd-free water or in concentrations equivalent to -5 or -1 1 % of LC50, and sampled after 2, 5, 8, and 1 1 wk. Total, dividing, and degrading or karyorrhectic cell numbers were recorded, as were cell length, one-sided surface area, and form factor. in fish exposed to -1 1 % LC50, total cell numbers and the incidence of cell division fell, while karyorrhexis increased, Dividing to total cell ratios declined with time; karyorrhectic ta total and karyorrhectic to dividing cell ratios increased. Variations in cytomorphology suggested slower cell maturation at both metal concentratians, Detection of heavy metal stress in fishes by this form of hematological assessment appears feasible.

On a 6tudie les modifications dans l'organisation des organes 6rythrocytaires chez le cyprin dore (Carassius auratus) expos6 pendant trois semaines i3 des concentrations equivalent i3 5, 15 et 25 % de la CL50 de 248 h du Cd. Au cours d'une etude ulterieure plus pouss&e, on a d'abord provoque une anemie s$v$re chez les sp4cirnens en les immergeant dans du chlorhydrate de phenylhydrazine, puis en les laissant se r4tablir dans une eau exempte de Cd ou A des concentrations equivalent approximativement 3 5 ou 11 % de la CL50; on a ensuite preleve des echantillons apr$s 2 , 5, 8 et 1 1 semi. Le nombre total de cellules ainsi que le nsmbre de cellules en voie de division et de cellules en caryorrhexie ou en voie de degradation ont 6tk notes, en plus de la longueur des eellules, de la surface erythrocytaire d'un seul c6t6 et du facteur morphologique. Chez les poissons exposes h une coneen- tration d'environ 1 1 % de la CL50, le nombre total de cellules et lrincidenee de la division eellkalaire dirninuent tandis que la caryorrhexie augmente. Le rapport entre le nombre de cellules en voie de division et le nombre total de cellules diminue en fonction du temps; cependant, il y a augmentation du rapport entre les cellkales en caryorrhexie et le nombre total de cellules ainsi que du rapport entre les cellules en caryorrhexie et les cellules en voie de division. Les variations cytomorphologiques laissent entrevoir u n ralentissement de la maturation cellulaire pour les deux concentrations de metal i3 l'etude. I I semble done possible d'evaluer le stress en presence de metaux lourds chez les poissons i3 I'aide de ce type d'evaluation hematologique.

Received December 7 9, 3 99 7 Accepted lu8y 7, 7 992 (JB342)

T he teleostean erythron is a dynamic entity whose ontogenic composition, and thus 0, transport capacity, reflects the interaction sf several processes. Prominent among

these are cell formation by pronephc and splenic erythropoiesis or peripheral cell division, maturation, aging. and cell breakdown or karyorrhexis (Murad et a%. 1990; Houston and Murad 1992). In complex multicomponent systems, factors that differentially influence the rates of the contributing activities inevitably alter overall status to some greater or lesser extent (Grodins 1963; Riggs 1963). This being the case, a pollutant exerting differential effects on any aspect of erythropoiesis an& or ontogenic progression would be expected to affect the composition of the circulating red cell population.

In the specific case of Cd, chronic exposure to nonacute environmental levels commonly leads to anemia (Johansson- Sjokck and Larsson 1978; Haux and Larsson 1984; Sjobeck et al. 1984; Gill and Pant 1985, 1986). This cannot be attkb- uted to hemodilution (McCarty and Houston 1976) but is well correlated with extensive lesisning of hematopoietic tissues (Stromberg et al. 1983) and impaired erytkopoietic capacity (Houston and Keen 1984). Detectable changes in erythron organization would therefore be anticipated at Cd concentrations below the acutely lethal. To test this hypothesis, red cell

populations of goldfish (Carassius auraaus) exposed under laboratory conditions to sublethal levels of Cd were periodically characterized for up to B 1 wk.

Materids and Methods

Origin and Maintenance of Goldfish

Goldfish (mean weight + 1 SEM 6.6 t 1.71 g) were obtained from a local commercial supplier (Hmz Mountain, Rexdale, Ontario). Upon arrival, they were surface disinfected by immersion in KMno4 and transferred for 2 wk to holding troughs maintained at 25.8 t 0.2"C, a widely accepted value for the thermal optimum of this species (HIouston 1982). Supplementary aeration maintained 0, levels at or above 6.8 mg/L (approximately 80% saturation). Photoperid hoods provided water surface light intensities of 90-120 lx on a 12 h light : 12 h dark photoperiod regime initiated at 08:80.

Animals were fed once daily to satiation on commercial pellets, a d excess food and fecal material was removed regulaly . As judged by general activity, feeding behavior, and the absence of obvious disease symptoms, they remained healthy throughout the study period.

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Experimental Protocol

The Cd content of dechlorinated St. Cathaines city water is relatively low (< 10 pg/L). Concentrations of the two other principal local heavy metal contaminants, Zn and Cu, are less than 58 pg/L and below reported threshold toxicities for most freshwater species. However, pH (7 .$-8 -0) and total hardness (135-150 mg/L as CaCO,) are such that added Cd is rapidly lost as CdCO,, Cd(OH),, and other precipitates. Earlier studies involving initial Cd loads of 1-60 mg/L, for example, revealed exponential decreases in dissolved Cd to negligible levels within 24-48 h (McCarty et d. 1978). Hardness and conductivity rose in proportion to the amount of Cd added, while total alkalinity and pH exhibited transient decreases. In the latter case, changes of as much as 1.8-1.2 pH unit were observed.

To avoid the complications inherent in time-variable conditions, water of reduced hardness was used. This was prepared by dilution of dechlorinated and aged city water with glass- distilled water and had the following characteristics: total hardness, 20-22 mg/L as CaCB, total alkalinity, 14-18 mg/L as CaCO,; conductivity, 15-25 siemens/cm; pH, 7 -4-7.8; [Cd2+], <2 ~ g l L . Little precipitation occurred in this medium following Cd addition, and changes in water quality were mark- edly reduced (McCarty et al. 1978).

In initial trials, fish were transferred in water to 100-L fibre- glass test tanks containing artificial soft water. Other conditions were as described. One week was provided for recovery h m transfer stresses. Analytical-grade CdC1, dissolved in glass- distilled water was then added to bring tank concentrations to 90, 270, and 445 pg/L, i.e. approximately 5, 15, and 25% of the softwater 240-h LC50 for this species (McC&y et al. 1978). Water levels were cchccked daily and adjusted to compensate for evaporation. Determinations by Beckman Spectrascan-5 flame emission spectrophotometry revealed little fluctuation with time. Following exposure to these conditions for 3 wk, dl specimens were sampled. This schedule was repeated to provide 10- 1 2 animals at each concentration.

A second, longer term study was then undertaken. Following acclimation to the conditions described, groups were brought to 5°C at a rate of -l0/d. To ensure comparability s f hematological status, all animals were rendered anemic by i m e r - sion in 1 mg/L solution of phenylhydrazine HC1 for 24 h in foil-shielded, aerated tanks (Houston et a. 1888). Following exposure, they were returned to their original tanks, treated with chlorstetracycline, and held at 5°C for 2 wk. Previous studies (Houston et al. 1988; Marinsky et al. 1990) have shown that this is sufficient to bring red cell numbers, hemoglobin, a d hematocrit to minimal values. Erythropoiesis was induced by increasing temperature to 25'C at a rate of 1-2"/d.

As in the original trial, it was intended to expose fish to -0, -5, and - 15% of LC50. Subsequent analyses revealed, however, that mean Cd concentrations were actually 84.5 md 1 87 pg/L, rather than 89 and 267 pg/L, i. e. 4.7 and 10.5% of the 240-h LC50. These are subsequently referred to as 5 and I Sampling was carried out after exposures of 2 ,5 , 8, and 11 wk and the schedule repeated to give the sample numbers subsequently reported.

To avoid possible induction of atifacts by chemical anes- thesia (Korcock et al. 1988), specimens were stunned with a spring-driven impact tool before sampling and killed i m e d i - ately afterward by trmsection of the medulla. Samples were drawn from the caudal vessels into dipotassium EDTA treated

collection tubes md held on ice before use. All hematological procedures have been described in detail (Houston 1990). Erythrocyte counts were carried out by hemcscytometer, Blood smears were prepared, air-dried, fixed in methanol, and stained with hishmmn-Giemsa. To estimate the abundmces of kar- yon-hectic and dividing cells, approximately 1000 cells were identified for each fish. Preliminary studies were carried out to determine the precision of the counting method. A minimum of four smears were prepared from a series of specimens. Sev- eral counts were carried out on each smear using a variety of methods for the selection of microscope fields. Results were then evaluated in terns of the effect of field selection method md number of replicate counts on mean and standard error. This indicated a minimum requirement of at least two smears per specimen and thee fields per slide selected from a pre- designated grid system using a random numbers table.

Cell deformation during smear prepartion can lead to distortion and difficulty in subsequent cell identification. Because of the particular sensitivity of thombocytes to physical distortion, only slides with thrombocytes of nomal morphology (Ellis 1977) were scored.

Categorization of the erythron on the basis of differential smear counting presents problems principally in relation to juvenile cells. As visualized by Romanowsky staining procedures, the nomal mature goldfish erythrocyte is symmetrically ellipsoidal. The ovoid, centrally positioned nucleus stains intensely and homogeneously blue-purple and the cytoplasm a pinkish orange. Nuclear chromatin and interchromatin regions and cytoplasmic inclusions are only occasionally seen. Recent studies have provided infomation on the cytomorphic characteristics of juvenile and mature red cells in this species (Hous- ton and Murad 199 1 ; Murad and Houston 1992). Thus, development of [%]thymidine-labelled cell cohorts in fish recovering from phenylhydrazine-induced anemia indicated that mature cells are characterized by mean major axis and one-sided surface area values, respectively, of 1 1.2 pm md 68.5 p2 or more. Axis ratio and form factor were 0.7 16 and 0.9 B 2 or less, respectively. Corresponding values for juvenile cells were major axis C9.2 pm; one-sided surface area r 5 0 pm2, axis ratio >0.774, form factor >0.938.

Thrombocytes , lymphocytes, monocytes, and the various granulocyte types c m be distinguished from mature erythrocytes on the basis of these variables, nuclear position, and nuclear morphology . While disagreement exists with respect to the classification of cells within this general category, the most common types can be differentiated from erythrocytes on the basis of cytoplasmic granulation md staining properties. Lym- phocytes, monmytes, and thrombocytes can usually be distinguished from juvenile red cells on the basis of their cytomorphic chatfacteristics, nuclear position, nucleus to cytoplasm ratio, md cytoplasmic staining properties. However, small numbers cannot be identified with certainty. These were recorded as uni- dentified cells and were not included in subsequent calculations.

In erythrocytes undergoing kqomhexis, cell and nuclear profiles typically shift from ovoid to round (Murad et al. 1990). Nuclear staining intensity is reduced and numerous irregular, vacuolelike structures are evident. Single or multiple irregular nuclear protmbmces are sometimes present. Cytoplasmic staining intensity is reduced and extensive vacuolization occuts. Enlargement of the nucleus is accompmied by reduction in cytoplasmic volume, and in the final stages of breakdown, only what appears to be a naked, swollen nucleus remains. Cells meeting these criteria do not incorporate radiothymidine.

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o - control ( < 2 pg Cdlb )

h w .- @ - 5% 240-k LC50 ( 85 yg C&L ) - aB r 0 - 11%24B-k hC50(187~gCd/L) 0 60 E

Exposure, d

FIG. 1. Accumulative mortality (% experimental population) in goldfish chonicdly exposed to -8% (<2 pg/L), -5% ($5 pg/&), and -11% (187 pg/L;B of 2404 Cd2' LC50.

Dividing cells display nuclear elongation with modest central construction (Mrad et al. 1990, 1992). This is followed by cellular elongation and constriction. Cell segments attenuate, move apart, and eventually separate. Their nuclei then shift centrally and cytoplasm volume increases. A taillike projection persists for an unknown period. Such cells take up both [%]thymidine and 55Fe (A. Murad and A. H. Houston, unpubl. obs .) .

Image analysis was employed to exmine the effects sf exposure to Cd on several cytomorphic variables. A Cohu Solid State 48 15-2088 camera linked to a Nikon 8 10326 microscope traced images within predefined shade limits, conveying the data to an IBM PCAT microcomputer. An Imaging Research Inc. software program translated trace data to values for cell and nuclear maximum chord (iength) and maximum perpen- dicular chord (width), one-sided surface area, and forrn factor (4.~-area/perimete?). Changes with time were taken as indic- ative of maturation rate. A minimum of 36 cells were measured from randomly selected fields on one randomly selected smear from each specimen.

Mortality

Postexposure mortality in phenylhydrazine-treated fish was 3%, well below the levels commonly encountered following intraperitoneal administration of, or immersion in, this hemolytic agent (Cameron and Wohlschlag 1969; Smith et al. 1971; Chudzik and Houston 1983; Houston et al. 1988). We believe that this may be attributable to the antibiotic treatment.

No mortalities were observed during initial 2 1 -d exposures to 90,270, and 445 pg Cd/L. Similarly, over the course of the longer term study, accumulative mortality in the control and 5% groups was Bow (Fig. 1). However, among fish exposed to 11% of LC50, mortality rose in near-exponential fashion after an initial lag period, accounting for 15% of the test group by week 11.

General Hematology

Prior to phenylhydrazine treatment, hematological indices were comparable with those previously re orted for this spe- ? ties, i.e. red cells 1.51 iz 0.107 x 10 /mm3, hemoglobin

C9

E r Dividing cells

Kayorrhectic cells s

Weeks FIG. 2. Total, dividing, md kqorrhectic red cells in goldfish chonically exposed to -0% (<2 pglE, open), -58 ($5 p,g/L, lightly cross-hatched), and -11% (187 pg/L, densely cross-hatched) of 24-h Cd2' EC50. Values reported as mean 2 1 s s ~ .

6.9 2 6.56 g/dL (Anthony 1961 ; Houston 1980; Murad et al. 1996). Separating cells were present but not common. At 7.18 k 1.89% of the total erythrocyte population, kqorrhectic cells were relatively abundant. Following exposure to phenylhydrazine, but prior to increase in temperature, red cell numbers had fallen to 0.12 iz 0.39 x 1 6 6 / m 3 (-92.1%). Kqorrhectic cells declined to 1.15 k 0.15% of the total, while separating cells accounted for 0.38 k 0.18% of the red cell population. Again, these were in reasonable agreement with earlier observations (Houston et al. 1988).

In the first test the incidence of kqonhexis was significantly elevated (P < 0.05 to P < 6.01) at all Cd concentrations, such cells comprising 0.36 + 0.5, 3.16 + 0.42, 1.29 & 0.39, and 1.30 + 8.18% of cells in the control, 5, 15, and 25% LC50 groups, respectively. Corresponding values for dividing cells were 0.34 5 0.06,0.38 + 0.07,0.36 k 0.05, and 0.45 ? 0.07%- Of these, only that from the 25% LC50 group differed significantly (P < 0.05) from the control group.

Variations during the subsequent, more prolonged study were consistent with those initially noted at comparable times (Fig. 2). As would be anticipated, total cell numbers increased

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Dividing / totai red cells Major axis, prn

Kayorrhectic /total red cells I

Karyorrhectic / dividing red cells

Weeks FIG. 3. Mean dividing to total, kqomhectic to total, and karyorrhectic to dividing cell ratios in goldfish chonically exposed to -0% (<2 kg/L, open), -5% ((85 pg/L, lightly cross-hatched), and - I 1 % (187 pg/L, densely cross-hatched) of 2 4 0 4 Cd2+ LC50.

significantly in all groups during the first 2-wk period. No significant differences between groups were evident. During the ensuing 9 k, the control and 5% groups did not change or differ significantly. By contrast, in the 11% group, cell numbers declined steadily and during weeks 8 md 11 fell significantly (P < 0-05) below those in both the control and 5% groups.

Much the same was true of the incidence of cell division. Dividing cell abundmces were elevated after 2 wk in both the control and 5% groups but exhibited no notable changes there- after. In the fish maintained at 11% LC50, their numbers fell to wear-negligible levels after 11 wk.

The occurrence of kqomhexis also differed with treatment. Although there was initially little difference among the test groups, kqorrhectic cell numbers increased steadily in the 1 1 % group and by the final sampling period were significantly in excess of those seen in the control and 5% groups.

Expressed as relative rather than absolute cell numbers, these differences were even more evident (Fig. 3). This was particularly the case with the 1 1 % goup. Thus, the ratio of dividing

One - sided surface area, prn2 B

Form fador

Weeks FIG. 4. Red cell major axis, one-sided surface area, and form factor in goldfish chronically exposed to -0% (<2 pg/L, opera), -5% (85 pg/L, lightly cross-hatched), and - 1 I % (1 187 pg/L, densely cross- hatched) of 240-h Cd2+ LC50. Data report& as mean f I SEM.

to total cell numbers fell from 0.033 after 2 wk to 0.005. Sim- ilarly, kqorrhectic cells relative to total cell numbers rose from an initial value sf 0.073 to 0.328 after 8 wk and 0.518 at the final sampling period. The mean ratio of kqsmhectic to dividing cells, which ranged from 4.6 to 10.7 in control and 5% animals, reached a final value of 1 12.6 in the 1 1 % group.

Cytomoqhology

Before induction of anemia, cytomovhic characteristics were similar to those previously reported for goldfish (Houston md Murad1992). Following treatment, but prior to exposure to Cd, mean cell major axis was reduced (Fig. 4). So was surface area, while f s m factor was sharply higher. Such characteristics are consistent with a population of preponderantly juvenile cells.

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Under Cd-free conditions, values for all variables increased steadily, reaching levels not significantly different from those of pretreatment fish after 11 wk. In both exposure groups, however, final values for major axis and surface area were well below those characterizing the control group. Similarly, mean form factor in both Cd groups exceeded that of control fish and, in the case of the 18% group, the difference was significant bP < 0.051.

As an approximation of cell age distribution, percentages of cells having major axes and surface areas less than and form factors greater than those observed before phenylhydrazine treatment were calculated (Table 1). In all groups, these pointed to steadily maturing cell populations. It will be evident, however, that control and test groups differed with respect to length and surface area during weeks 8 and 11. Such values suggest some retardation of the maturation process.

Material flow through aquatic food chains commonly leads to bioconcentration at higher trophic levels. Because of this, and the intimacy of aquatic organism - environment interac- tions, the occupants of these levels can be regarded, in some respects at least, as integrators of habitat conditions. This being the case, it has been suggested that development of reliable means for the detection and quantification of stress states in fishes might provide a basis for assessment of overall environmental quality. Beitinger and McCauley (1990) and Wedemeyer et al. (1990) have recently reviewed the growing literature in this field, systematizing and evaluating approaches to stress assessment in terms of their biochemical, physiological, and organismic manifestations.

Responses to stress are commonly termed primary, secondary, and tertiary. Of these, primary responses stem from direct stressor effects at the cell molecular level, e.g. on membrane organization and permeability, substrate activation state, the activities of specific enzyme systems, etc. Induction of endo- crine responses is also regarded as primary although these responses presumably reflect more primary events at the cellular level. Secondary responses stem from primary effects and are often seen as deviations from the norm in various regulated activities: osmotic, ionic, and acid-base regulation, gluco- stasis, immunological competence, erythron status, acclima- tory capacity, and similar functions. Effects at the tertiary or whole-organism level include a wide range of activities such as overall metabolism, growth, competitive and reproductive abilities, mortality, etc.

The hematological changes observed in stressed fish presumably reflect more primary events such as stressor-induced variations in specific enzyme activities and circulating levels of catecholmines, steroids, and other stress-related hormones,

i.e. they are derivative or secondary responses. As is the case with many such effects, the pathway(§) by which pri are translated to detectable responses are not well understood for the most pat . Nevertheless, their occurrence is undoubted.

In concept, the utilization of hematological variables for stress detection is attractive. Unit costs for the determination of the common primary and secondary indices (red and white cell counts, hematocrit and leucocrit , total hemoglobin, mean erythrocyte volume, and hemoglobin) are re la t idy low, while through-put is normally high. The procedures involved do not require unusual facilities or expertise. However, as currently employed, they are insensitive. The changes most commonly reported (anemia, polycythemia, leucopenia, and leucophilia) are frequently seen only under circumstances of acute response to gross challenge (Wedemeyer et al. 1990; Ssrensen 199 1).

Nevertheless, recent studies indicate that the blood cell system offers scope for more subtle employment. Once regarded as relatively stable, the erythron is emerging as a dynamic, responsive entity (Houston 1990). Respiratory stresses as diverse as reduced blood 0,-carrying capacity, transient exposure to hypoxic conditions, and transient thermal elevation of 0, demand invoke a common pattern of response. This is characterized by increases in rates of eqthropoiesis, cell maturation, division of circulating juvenile cells, and karyorrhexis (Murad et d. 1990; Houston and Murad 1992). As a result the age distribution of the red cell population shifts in favor of immature cells. This is critically important. Only two factors can be adjusted at this level: hemoglobin-0, affinity md blood 0,-caving capacity (Houston 1980; Powers 1980; Weber 1983). These are a function of (1) the types and amounts of specific hemoglobin isomorphs present md (2) cell levels of affinity-modulating solutes such as ATP, GTP, H' , C1- and h4g2 ' . In turn, these appear to be related to the conditions under which juvenile cells mature (Byrne and Houston 1988; Mar- hsky et al. 1990). The coupling of new cell addition to elim- ination of some proportion of the preexisting cell population permits significant alteration in overall blood gas transport characteristics. In addition, a system of this kind offers the advantage of minimizing increases in overall cell numbers, and therefore viscosity and ultimately cardiac work requirements.

Our observations support the prediction that detectable, significant changes in eqthron organization follow long-term exposure to Cd concentrations well below acutely lethal levels. Indeed, even total red cell count, a relatively insensitive index, was altered after 8-1 1 wk of exposure to only 10% of the 240-h LC50.

A number of factors may have contributed to observed changes. At any given moment the number of circulating red cells represents a balance between all processes adding cells to or removing them from circulation. The lymphomyeloid tissues of the pronephros and spleen are principal eqthropoietic sites

TABLE 1 . Relative abundances s f erythrocytes having major axis ( 1 1.2 pm), one-sided surface area (68.3 p,rn2), and form factor (0.9 19) values less than pretreatment values.

Major axis Surface area Form factor

Time Control 5% '0 '0% Control 5% 1 1 % Control 5% 11%

"Fish sampled 2 wk following exposure to phenylhydrazine HCl.

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of teleosts (Fange 1986). The damage to these tissues observed by Stmmberg et al. (1983) and the inhibition of heme synthesis reported by Johmssow-Sjobeck and Lasson (1 978) presumably underlie demonstrated reductions in erythropoietic capability (Houston and Keen 1984).

Division of circulating cells was also significantly reduced. Apparently limited to immature erythrocytes (those possessing the organelles required for hemoglobin synthesis), division leads to cells able to take up and incorporate "Fe into hemoglobin (Mwad et al. 1992). En addition, however, k q o h e x i s dso increased significantly. Consequently, it would seem reasonable to attribute observed decreases in cell numbers to reduced red cell formation and removal of existing, presumably senescent cells through increased kqonhexis .

However, the effects of Cd were more obvious in relation to dividing to total, kqomhectic to total, and particularly kar- yomhectic to dividing cell ratios. All were m k e d l y altered during even the relatively short-duration exposures employed in this study. Moreover, since essentially all heavy metal pol- lutants affect heme metabolism, cytoskeletd organization, and cell division and/or exert other rate-controlling or toxic effects (Sorensen 199 11, these simple, eomon ly used hematologicd procedures may be of general utility.

It is worth noting in this context that changes in the absolute and relative abundmces of leucoeytes also follow exposure. Under generally similar conditions, total Heucocyte numbers decreasedfi-om4.92 5 0.72 x 1O3t022.52 * 2.32 x 10" cells/mm3 after 3 wk at 225% LC58 (Murad and Houston 1988). Thrombocyte numbers remained approximately constant. while lymphocyte abundmces declined and granulocyte numbers increased. As a result, the mean neutrophil t s lymphocyte ratio rose more than fivefold from 0.087 to 0.478. Clearly, these as well as erythron variables may be useful indicators sf stress.

Finally, since the erythrocyte md leucocyte variables exhib- iting the largest changes are all ratios, blood smears prepared in the field by minimally traumatic sampling procedures for subsequent evaluation in the laboratory may suffice for diag- nostic purposes.

The authors gratefully acknowledge the financial support provided by the Natural Sciences and Engineering R e s e m h Council s f Canada though Ind. Op. Grmt A6972 to A. H. Houston. Dr, R. W. McCauley provided valuable comment on the manuscript.

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