564

Our results could have been biased in several ways.First, the influence of changes in body-surface areashould be considered, since variables were converted toa standard body-surface area of 1-73 sq.m. In four

patients an increase of body-surface area by 0-08-0-11 sq.m. was calculated, due to weight gain. Changesin hxmodynamic variables, converted to standard

body-surface area, could not be accounted for by thisslight increase in body surface. Secondly, the reduc-tion of hxmodynamic variables in the follow-up studymight be due to psychological acclimatisation to theinvestigative procedure. Although some influence ofthis kind cannot be ruled out, it is most unlikely thatit has been a decisive one, for the following reasons.We found no proportional decrease in blood-pressure,as is apparent from calculated values of peripheralresistance; the changes in renal blood-flow cannot beexplained on a psychological basis, since renal blood-flow tends to be reduced during psychic stress, whilein fact changes roughly paralleled cardiac output(fig. 2); and we found no evidence of a reduction incardiac output during short-term repeat studies (inter-val 1-2 weeks), so conditioning to the procedure doesnot seem to be important. We conclude that our datarepresent pathophysiological changes in the course ofhypertensive disease. Individual decreases in cardiac

output and renal blood-flow were excessive in com-

parison with the general decline of values along thescale of increasing age. Hypertension, as a matter offact, may start at any age and the development ofhypertensive disease takes less than a lifetime.

In contrast to the rather homogeneous pattern ofdecreases in cardiac output and renal blood-flow,blood-pressure and blood volume developed in anirregular fashion.The observation that blood-pressure fell in some

patients should not be interpreted simply as a " lapse "of hypertension, since total peripheral resistance orrenal vascular resistance in some cases actually in-creased to more pathological values.

Patients with a myocardial infarction more or lessfell in line with the other patients, although two ofthem showed an exceptionally steep drop in blood-pressure. The measurements in this group may serveas a useful reference for other observations. After

myocardial infarction, the mechanism of haemodynamicalterations is identifiable as a decrease in myocardialcontractile force. The sequence of events would seemto be as follows: cardiac output drops, entailing areduction of renal blood-flow; blood-pressure falls inthe wake of cardiac output, but it does not decrease to a

proportional degree, as is indicated by an increase intotal peripheral resistance which may be ascribed to anintervention by the baroreceptors. In most uncompli-cated cases a decrease in blood-pressure may also besupposed to result from reduction of myocardial per-formance. In some of these a decrease in blood volumewas demonstrated, and this could be the underlyingmechanism. In those cases where the blood volumedid not fall, the mechanism becomes entirely specula-tive : some neurovegetative change or structural

damage may have occurred in the myocardium. Themain difficulty in interpreting these data is that wecould find no relationship between changes in blood-pressure, cardiac output, and blood volume.

In future follow-up studies, therefore, we wouldstrongly advocate serial measurements of other vari-ables, such as plasma-renin and catecholamine secre-tion.

We thank Miss B. L. Spildooren, Miss A. T. Edixhoven,Miss E. M. Enis, Miss J. E. Schot, and Mr. L. Ries for technicaland administrative assistance, and Dr. J. Geerling for adviceand criticism. Investigations were supported by grant G.O.683-61 from the Dutch Organisation for Health Research

T.N.O., and by a grant from the Kidney Foundation in theNetherlands.

Requests for reprints should be addressed to W. H. B.

REFERENCES

1. Stewart, I. McD. Lancet, 1971, i, 355.2. Levy, R. L., Hillman, C. C., Stroud, W. D., White, P. D. J. Am.

med. Ass. 1944, 126, 829.3. Birkenhäger, W. H., Van Es, L. A., Houwing, A., Lamers, H. J.,

Mulder, A. H. Clin. Sci. 1968, 35, 445.4. Bello, C. T., Sevy, R. W., Harakal, C., Hillyer, P. M. Am. J. med.

Sci. 1967, 253, 194.5. Eich, R. H., Cuddy, R. P., Smulyan, H., Lyons, R. H. Circulation,

1966, 34, 299.6. Lund-Johanson, P. Acta med. scand. 1967, 181, suppl. no. 482.7. Schalekamp, M. A. D. H., Schalekamp-Kuyken, M. P. A., Birken-

hager, W. H. Clin. Sci. 1970, 38, 101.8. Sannerstedt, R., Schröder, G., Werkö, L. Acta med. scand. 1971,

190, 419.9. Reubi, F. C. in Essential Hypertension (edited by K. D. Bock and

P. T. Cottier); p. 317. Berne, 1960.

ALUMINIUM TOXICITY IN RATS

G. M. BERLYNE

J. BEN ARIE. KNOPF

R. YAGIL

G. WEINBERGER

G. M. DANOVITCH

Department of Nephrology, Negev Central Hospital, andDivision of Life Sciences, Negev Arid Zone Research Institute,

and Faculty of Natural Science, University of Negev,Beer Sheva, Israel

Summary Aluminium intoxication has been dem-onstrated in the uræmic and non-uræmic

rat after modest doses of oral and parenteral aluminiumsalts. The clinical syndrome is associated with peri-orbital bleeding, lethargy, anorexia, and death. Plasma-levels of aluminium were greatly raised, as were tissuelevels in liver, heart, striated muscle, brain, and bone.Histological changes were found in the cornea. Liver

oxygen consumption was reduced by giving theanimals aluminium salts before death or by addingaluminium in vitro to normal liver homogenates. It isrecommended that aluminium salts should be with-drawn from use in patients with renal failure andtheir use restricted in normal persons pending clarifi-cation of the issue.

Introduction

THE question-is aluminium toxic ?-has never

been answered satisfactorily. 1- We have found thatnormal rats and rats in renal failure develop fatalintoxication from modest doses of aluminium salts;plasma-aluminium levels and tissue concentrationswere increased, and the addition of aluminium to

liver homogenates in vitro depressed oxygen uptake.

565

Materials and Methods

Littermate white adult male rats of Weizmann Institutestrain weighing 200 g. were divided into control groupsand test groups. The following test procedures weredone (table r).

5/6 NephrectomyA total nephrectomy was carried out on one side and a

2/3 nephrectomy was done on the other side under chloralanaesthesia. The animals were allowed to recover for 1-2weeks from the operation and were then divided intocontrol and test groups. Both groups were given standardrat pellets as food.

Aluminium SaltsTest groups were given 1 % or 2 % aluminium chloride

(AICla) or sulphate (AliS04)a) as drinking-water or oralaluminium hydroxide (AI(OH)3) 3 ml. daily (equivalent to150 mg. of elemental aluminium per kg. per day) bygavage. Further test groups were given subcutaneous orintraperitoneal AI(OH)a in a dose of 3 ml. per day (equiva-lent to 90 mg. per kg. body-weight per day). All experi-ments were controlled with animals which had undergone5/6 nephrectomy but received tap-water in place of alu-

TABLE I-DESIGN OF IN-VIVO TOXICITY, INCLUDINGROUTE OF ADMINISTRATION AND MORTALITY

minium salts to drink. In the experiments with intra-peritoneal or subcutaneous injections, sham injections werenot given to controls.

Normal RatsGroups of non-operated (non-nephrectomised) rats

were given 1% AJCIg and AI2(S04)a as drinking-water, andAl(OH)s by gavage and intraperitoneal and subcutaneousroutes. Control rats were given distilled water in place ofaluminium salts.

Oxygen Consumption in VitroThis was determined in liver homogenates in a Warburg

apparatus, using liver from non-nephrectomised animalsgiven oral or intraperitoneal Al(OH) and normal rats drink-ing water as controls. Total protein was measured by theLowrey technique and results were expressed as pd.oxygen consumed per minute per mg. protein. The testwas repeated using normal homogenate of healthy rat

liver to which A12(S04)3 was added in various concentra-tions up to 40 mg. per litre and oxygen consumption com-

Fig. I-Periorbital bleeding 6 days after one injection of intra-peritoneal AI(OH)3 in a non-operated rat.

pared with controls. Buffer used in all instances was

Krebs-Ringer-phosphate buffer pH 7-4.

Tissue LevelsAluminium in tissues was extracted after drying in

’Pyrex’ tubes at 100°C for 4-7 days and dissolving theresidue in concentrated hydrochloric acid for 1 week.Aluminium was measured on a Unicam SP90 atomic-absorption spectrophotometer. (Heating at 6000C in aporcelain crucible gave spurious increases in aluminiumbecause aluminium was leached out of ceramic crucibles,which contain 2% aluminium oxide.)

Results

Clinical Picture

5/6 nephrectomised controls recovered rapidly fromthe operation, and none died within 3 weeks of opera-tion, whereas all animals receiving 1 % of Aids orAl2(SO 4) died within 8 days and all receiving 2% A1C13or Al2(S04)a died within 3 days. The clinical picturewas lethargy, failure to thrive, bleeding around the eyes(fig. 1), and reduced spontaneous movement. The testrats ate and drank very little in the last 2 days of life.Rats on 2% aluminium salts drank 11 (range 5-15) ml.water per day over 4 days, but intake on the first 2 dayswas probably around 20 ml. per day. They did not dieof ursemia, the heart-blood urea being 320 mg. per100 ml. at death. Rats on 1 % aluminium salts drank18 (range 16-21) ml. per day during 8-9 days’ survival.Rats given aluminium intraperitoneally or subcu-

taneously and the control rats on pure water all drank26-30 ml. per day. To exclude the possibility that thedeaths were due in some measure to acidosis or

hypophosphatxn-lia caused by oral aluminium salts, orto dehydration caused by voluntary limitation of fluidintake, other groups of animals were given 30 mg. ofelemental aluminium as aluminium hydroxide in asingle injection intraperitoneally or subcutaneously.Similar results with a high mortality within 5 dayswere seen in nephrectomised and control animals

given intraperitoneal injections of Al(OH)a. Subcu-taneous injections were less toxic, resulting in nomortality, but periorbital bleeding occurred in nephrec-tomised animals. In non-operated animals 2%AliS04)a caused periorbital bleeding in 3 out of 5rats, but no mortality.

Histological FindingsAnimals which died with periorbital bleeding had

566

Fig. 2-Control cornea showing mitotic figures in stratumbasale of epithelium and wide epithelial surface.

( x 250.)

Fig. 3-Cornea of animal given intraperitoneal Al(OH)a 5 dayspreviously.Note lack of mitoses in stratum basale and narrowed epithelial

layer. (x 250.)

conjunctival epithelial ulceration in the fornicealconjunctiva, and the bulbar conjunctiva was reducedin thickness compared with controls, with a strikinglack of dense mitotic nuclei in the stratum basale ofthe corneal epithelium (figs. 2 and 3). The lungsshowed a histological appearance of patchy pneu-monia with desquamation of bronchial epithelium.The liver was normal apart from perihepatic aluminiumgranulomas and fibrinous peritonitis in animals givenintraperitoneal aluminium hydroxide. The heart,spleen, pancreas, and skeletal muscle seemed normal.Histological examination of bone is not yet complete.

Biochemical Findings (tables II-IV)The highest tissue concentrations in controls were

found in bone (table 11). In rats given intraperitonealAl(OH)s the tissue concentration was highest in theliver (table ill), but the concentrations for muscle,brain, bone, and heart were also several hundredfoldhigher than for the controls. 5/6 nephrectomisedanimals given oral Al2(SO 4) consumed an average of18 mg. elemental aluminium; the highest levels arefound in bone, but the levels in liver, heart, andmuscle are also considerably increased in rats survivingto the 8th day (table iv). The serum-inorganic-

TABLE II-ALUMINIUM CONTENT OF NORMAL RAT TISSUES:

MEAN OF 5 RATS, POOLED TISSUES

TABLE III-TISSUE LEVELS OF ALUMINIUM AFTER

INTRAPERITONEAL Al(OH).: 5 RATS, MEAN OR MEAN AND RANGE

TABLE IV-EFFECT OF ORAL 1% A12(S04)3 ONTISSUE LEVELS OF ALUMINIUM (GROUPS OF 5 RATS)

All animals died within 8 days of starting aluminium-salt drinking

phosphorus level in animals dying after parenteralA1(OH)9 was 10-5 mg. per 100 ml. (mean of five rats).Aluminium concentrations were very high in brain,but were raised in all tissues (table iv).

Serum-levels of aluminium were high in all groupsof test animals (table v), the highest figures (39-4 mg.per litre) being found in nephrectomised animals onoral 2% Al2(S04)3’

Oxygen consumption fell by 25% in aluminium-treated rats (table vi). In-vitro addition of aluminiumto healthy rat liver led to an immediate depression ofoxygen consumption, but this fell short of statisticalsignificance.

TABLE V-EFFECT OF SUBCUTANEOUS ADMINISTRATION OF

Al(OH)3 ON TISSUE ALUMINIUM LEVELS: MEAN AND RANGE

TABLE VI-SERUM-ALUMINIUM LEVELS: 5 RATS IN EACH GROUP

567

Discussion

Aluminium toxicity has been the subject of sporadicinvestigations in the past twenty years. 3,9 Aluminiumhas been administered principally in the form of oralAl(OH) in the rat, in which a phosphorus-depletionsyndrome is induced with depletion of red-cell A.T.P.3,9 9This should not be primarily considered as a mani-festation of aluminium toxicity, but rather as a result ofphosphate depletion resulting from phosphate bindingin the bowel. A similar syndrome has been reportedin man caused by mixed Al(OH) and magnesium oxideintake.’ In normal man absorbed aluminium is readilyexcreted by the intact kidney. It is likely, therefore,that oral administration of aluminium salts will resultin toxic manifestations in patients with renal failure,for raised plasma-aluminium levels have been foundafter aluminium salt or aluminium resin intake. 8 It isin these patients that a situation analogous to thealuminium toxicity of the 5/6 nephrectomised rat mayoccur if aluminium intake is increased by aluminiumresins or other absorbed aluminium salts. The lack ofobservations on the toxic effects of aluminium (otherthan phosphate depletion) is possibly the result of

giving poorly absorbed aluminium salts orally to ratswith normal renal function. The combination ofaluminium salts orally or parenterally to uraemic rats ispresumably responsible for the high morbidity andmortality rates in our experiments. The minimumfatal dose in the rats given oral sulphate or chloridewas about 1100 mg. elemental aluminium per kg. body-weight ; in rats given intraperitoneal salts the dosechosen (150 mg. per kg. per day) was fatal in all rats.

In our experiments aluminium toxicity was moststriking in ursemic rats, and manifested in a clinicalsyndrome of lethargy, periorbital bleeding, anorexia,and death. Plasma levels (normal value 0-2 mg. perlitre) were raised to between 0-5 and 40 mg. per litre,depending on the route of administration of the alu-minium salt. These levels correspond with those foundin patients in renal failure taking aluminium resins orAl(OH) 3 therapeutically. 8 Although aluminium intoxi-cation could be induced most readily in ursemic rats,toxicity could also be induced in normal rats by oralor intraperitoneal aluminium salts. The oral dose

given to uraemic rats of 18 mg. elemental aluminiumper day (90 mg. per kg. body-weight) is two or threetimes the dose given therapeutically to patients inrenal failure, and about equal to the amount of alu-minium theoretically available from aluminium resin ifresin is taken in a dose of 45-60 g. per day. Since, atleast for the urxmic rat, aluminium seems to be toxicin a dose close to or identical with the therapeuticdose used in ursemic man, we recommend that, pend-ing further work, aluminium salts should not beprescribed for patients with renal functional impair-ment, and that they should be withdrawn from themarket. A wide range of aluminium salts is used-they should not be regarded as inert.The massive deposition of aluminium in various

organs-notably, brain, liver, heart, and muscleindicate that aluminium may have some responsibilityin the production of several little understood uraemicsyndromes, especially where bone is concerned. Wehave found aluminium levels, in samples of bonepowder supplied by Dr. M. Kaye from uraemic

TABLE VII-IN-VITRO OXYGEN UPTAKE IN LIVER

HOMOGENATES OF RATS

Group l.-Treated with AI(OH)g intraperitoneally, and control group.Group 2.-All livers from normal (untreated) rats. 40 {ig. per ml.aluminium as Al2(SO.). added to homogenate at start of incubation,nothing added to controls.

t Significantly lower than control (t = 2-78, P<0’05).: Not significantly lower than control (t = 2-42, 01 > r > 0-05).

men not known to have taken aluminium salts, tobe twice those in non-ursemic men (unpublished),a result which accords well with that of Parsonset all who also found a large amount of aluminiumin urasmic bones. The implication of aluminiumsalts in the xtiology of the metatarsal type of osteo-porosis seen in Newcastle is suggestive, in viewof the high Al content of bone in Newcastle. Wehave suggested 5 that aluminium may damage boneformation, and this accords with the evidence ofBachra and van Harskamp 6 that 1 mg. of aluminiumcan initiate hydroxyapatite precipitation.The action of aluminium salts in vitro in depressing

liver respiration is indicative of a direct toxic action onthe liver cell. Further work is being done to determinethe intracellular level and mechanism of aluminiumpoisoning. There is a reduction in liver-proteinconcentration which may be due to direct interferencewith protein synthesis, being found in non-urasmic ratsdrinking Al(OH) 3.The previous work on aluminium toxicity has to a

certain extent been befogged by the use of oral routesof administration of aluminium salts and rats withnormal renal function. This allows phosphate deple-tion to develop and is responsible for the incorrectassumption that aluminium toxicity is really phos-phate depletion. That this is not so is demonstrated

by slightly raised levels of phosphate in the uraemic ratwith aluminium poisoning, in which phosphate deple-tion can be confidently excluded.The reduction in mitoses in the stratum basale of the

corneal epithelium is presumably a response to severealuminium intoxication. Periorbital bleeding is a veryregular finding in the non-urxmic rat given intra-peritoneal aluminium hydroxide, and may well be dueto damage to the forniceal conjunctiva.

Although it is difficult to extrapolate from the ratto man it would seem advisable to suspend the wide-spread use of aluminium salts in man pending furtherstudies of this problem.We thank Dr. H. Doron and Mr. M. Soroka, of Mercaz

Kupat Holim, Tel-Aviv, for making this work possible.Requests for reprints should be addressed to G. M. B., Depart-

ment of Nephrology, Negev Central Hospital, P.O. Box 151,Beer Sheva, Israel.

REFERENCES

1. Berlyne, G. M. Lancet, 1970, ii, 1253.2. Wrong, O. M. ibid. p. 1130.3. Ondreicka, R., Ginter, E., Kortus, J. Br. J. ind. Med. 1966, 23, 305.

568

4. Parsons, E., Davies, C., Goode, C., Ogg, C., Siddiqui, J. Br. med. J.1971, iii, 273.

5. Berlyne, G. M., Yagil, R., Weinberger, G., Poplicker, F. in Ur&aelig;mia(edited by R. Kluthe and G. M. Berlyne). Stuttgart (in the press).

6. Bachra, B. N., van Harskamp, G. A. Calc. Tissue Res. 1970, 4, 359.7. Lotz, M., Zisman, E., Bartter, F. C. New Engl. J. Med. 1968, 278,

409.8. Berlyne, G. M., Ben Ari, J., Pest, D., Weinberger, J., Stern, M.,

Gilmore, G. R., Levine, R. Lancet, 1970, ii, 494.9. Poole, J. W., Zeigler, L. A., Dugan, M. A. J. pharm. Sci. 1965,

54, 651.

STERILITY AND TESTICULAR ATROPHYRELATED TO

CYCLOPHOSPHAMIDE THERAPY

K. F. FAIRLEY JEAN U. BARRIEWARREN JOHNSON

Renal Unit of the Royal Melbourne Hospital and theRoyal Women’s Hospital, Melbourne

Summary All samples of seminal fluid from

thirty-one adult male patients who hadreceived cyclophosphamide showed low sperm-countsor azoospermia. Studies showed that the sperm-countcan fall to zero after 4 months’ treatment, and all

patients who had been treated for 6 months or moreshowed azoospermia. Only two out of ten patientswho were studied 3-19 months after stopping cyclo-phosphamide showed any mature spermatozoa in theseminal fluid, and the counts in both these patientswere very low. Preliminary studies suggest that

cyclophosphamide may also cause ovarian damage.

Introduction

ALTHOUGH cyclophosphamide was originally usedfor the control of malignant conditions it is now widelyused for the treatment of the nephrotic syndrome,particularly in childhood,1-7 where controlled trialshave established its undoubted benefit. 6 Sterility inone of our patients after a course of cyclophosphamidecaused us to investigate the time sequence of changesin the sperm-counts of men receiving cyclophospha-mide.

Material and Methods

Fresh samples of seminal fluid from 31 adult patientswho had received cyclophosphamide were examined, andspermatozoa were counted using a standard counting-chamber. Sperm-motility was studied by the eosin-

staining method. If examination of one hundred high-power fields failed to find any spermatozoa the centrifugeddeposit was examined to confirm azoospermia.

Testicular biopsy was carried out in five patients aged24-44 years. Three patients had stopped cyclophosphamide1 month, 12 months, and 15 months before the biopsy.The other two were on treatment at the time of the biopsy.In all patients cyclophosphamide was given in a dose of50-100 mg. per day. Renal function was normal or onlyslightly impaired in all patients (serum-creatinine less than2-0 mg. per 100 ml.).

Results

Eighteen patients who had received cyclophospha-mide for 6 months or more, and were on treatment atthe time of the examination, showed azoospermia.Eleven patients who had received cyclophosphamide

SEMEN ANALYSIS IN ELEVEN PATIENTS 3-19 MONTHS AFTER WITH-DRAWAL OF CYCLOPHOSPHAMIDE

for 2 months or more had semen analysis performedat different intervals after stopping cyclophosphamide.Only three patients had any mature spermatozoa, andthese counts were very low (see accompanying table).

Serial counts were carried out in six patients, 1 weekto 6 months after the start of cyclophosphamide treat-ment. In some patients the sperm-count was reduced3 weeks after starting treatment. No patient developedazoospermia unless at least 4 months’ treatment hadbeen given. In two patients sperm-counts continuedto fall for 2 to 3 months after treatment finished. Fig. 1shows the effect of 3 weeks’ treatment.

Testicular biopsy was performed on five patients,and no spermatogenesis was found in patients whowere receiving cyclophosphamide at the time of thebiopsy or in the patient who had stopped treatment6 weeks before the biopsy. One patient showed onlyoccasional spermatogonia in the testicular tubules

despite an interval of 12 months after stopping cyclo-phosphamide. Most tubules contained only Sertolicells (fig. 2). Spermatogenesis was seen in a testicular

Fig. 1-Rate of fall of absolute sperm-count and percentagemotility during treatment with cyclophosphamide.The first count was done during the third week of cyclo-

phosphamide (100 mg. per day). This treatment was stopped for3 months, but the sperm-count continued to fall for 10 weeksafter cyclophosphamide was discontinued. Treatment was thenresumed.