toxicological consequences of chloroquine and ethanol on the developing fetus

Pharmacology Btochem,stry & Behavtor, Vol 34, pp 77-82 © Pergamon Press plc, 1989 Planted m the U S A 0091-3057/89 $3 00 + 00

Toxicological Consequences of Chloroquine and Ethanol on

the Developing Fetus

A R C H A N A S H A R M A A N D A R U N K R A W A T 1

Mzdwest lnstttute for Treatment and Study of Alcohohsm Universtty of Toledo, P 0 Box 5888, Toledo, OH 43613

Rece tved 28 D e c e m b e r 1988

SHARMA, A AND A K RAWAT Toxwologwal consequences of chloroqume and ethanol on the developmg fetus PHARMACOL BIOCHEM BEHAV 34(1) 77--82, 1989 --In the present study, the effects of chloroqume and ethanol adnumstratlon dunng gestation have been mvestagated on the developing rat fetus Intragastnc adnumstranon of chloroqulne (700 mg/kg body weight) resulted in several structural abnormalltaes The incidence of hepatomegaly was increased by 30%, the hqulfication of visceral organs was increased by 15% and a 9% higher incidence of cleft palate, wrist drop, clubbed foot and brain hqulficataon was observed m the fetuses from the chloroqume-treated group compared to the corresponding controls Fetuses from the chloroqume-treated group also showed a decrease of about 40% in the body weight and a 30% reduction in the ossification of the sternum The teratogemc effects of oral ethanol adnumstratlon in several respects were similar to those of the chloroqume Ethanol, when administered as 30% of the total daily calories, resulted in growth retardation, resorption, still births, hqulficatlon of the brain, wrist drop and clubbed foot Additionally, ethanol resulted in the inhibition of several metabohc pathways in the liver and brain of the developing fetuses This included the mMbltlon of protein, RNA and DNA metabolism in the fetal livers and brains The feto-toxlc effects of these two xenoblotlcs and their possible molecular mechanisms have been discussed

Chloroqume Ethanol Fetus Hepatomegaly Cleft palate Brain hqulficatlon Inhibition of RNA DNA Protein metabolism

IT is now well estabhshed that a variety of xenoblotlcs, alcohols and drugs cross from the maternal ctrculatton to the developing fetus via the placenta in both humans (1,3) and laboratory animals (5, 8, 12, 13) Highly hpld soluble xenoblotlcs cross the placenta m amounts that are proportional to the maternal-placental blood flow (5) Furthermore, a large number of drugs admlmstered to the lactating mothers are detectable in the maternal milk. During early gestataon, particularly at the time of organogenesls, the fetus ~s highly susceptible to the toxic and teratologlcal effects of the drugs For some time now chloroqulne has been not only deployed as a potent antimalarial agent, it has also been used in the treatment of mflammauon and arthritis in recent years Although in the past decade other antimalarial drugs such as quinine (11,15), prynmethamme and paludnne (14) have been shown to exert terotolog~cal effects in species such as rabbits, guinea pigs, hamsters and rats, the teratologlcal potential of chloroqume has not been established eqmvocally (2) On the other hand, the teratogenlc potential of drugs hke ethanol is now well established in the precllnlcal studies with animals and in humans (3, 6, 10) In human fetus and offspnng from chronically alcohol abusing

mothers, an array of anatomical, behavioral and metabolic abnormalities have been observed (3,10) The con&tlon has been called Fetal Alcohol Syndrome

The present mvestlgatlon ts undertaken to investigate 1) the potential teratogemc effect of chloroqume, 2) to investigate the differences and stmtlantles between the teratogentc effects of chloroqutne and ethanol, and finally 3) to investigate the pharmacological correlates of the teratogemc effects of ethanol on the developing fetus

METHOD

Chemwals (Ul4C)alanme, (14C)valine, (14C)leucme, (14C)thymldme, (5-

3H)undme and other radlochemlcals were purchased from New England Nuclear Corp (Boston, MA) Enzymes and other bto- chemicals were obtained from Boehnnger Mannhelm Corp (New York, NY) All other chelmcals were of reagent grade Alazann red, hematoxyhn and DPX mount were products of Aldrich Chemmal Co Chloroqume &phosphate was obtained from Rouche Pharmaceutical Co Ltqmd Sustacal was obtained from Mead

~Requests for repnnts should be addressed to Arun K Rawat

77

78 SHARMA AND RAWAT

TABLE 1

HIGH INCIDENCE TERATOGENIC EFFECTS OF CHLOROQUINE

Types of Abnonnalmes Treatment Abnormalities % of Control

Chloroqulne (700 mg/kg) Reduction m size 46 0 (76) for 15 days Hepatomegaly 21 1 (29)

Llqmficatlon of 15 0 (43) visceral organs

Wavy ribs 6 0 (36) Sternum 12 0 (47) Upper &glts 100 0 (47) Lower &glts 100 0 (47) Sacral 4 5 (47)

Effects of prolonged chloroqume admmlstraUon during gestation have been stu&ed on the developing fetus compared to the corresponding fetuses from the sahne-treated group H~gh incidences of teratogemc effects were observed by using macroscopic, shcmg and skeletal stu&es as descnbed m the Method section The results are expressed as percent of controls w~th the number of observations m parentheses N D denotes not detectable

Johnson C o , Evanswlle, IN The details of liquid d~et preparation and its nutritional components have been described previously (7)

Animals

Albino Wlstar rats were maintained on laboratory chow and water ad hb Female rats with body weights of 200-250 g and males of 300-350 g were mated for 12 hr in the ratio of 3 1 (male female) The presence of sperm m the microscopic examination was taken as an evidence of mating and was counted as day zero of pregnancy The ammals were &vlded into control and experimental groups Chloroqulne was admlmstered lntragastrl- cally (700 mg/kg/body weight) The control group recelved equal volumes of 1 N saline Ethanol was gwen m hquld Sustacal &et as 30% of dally &etary calones The corresponding control group was pmr-fed on lsocalonc Sustacal-sucrose hquld diet The fetuses were removed at defimte intervals of treatment m all cases and were employed m different anatomical and biochemical studies

Preparatton of Ttssue for Teratologtcal Studies

To study the teratogenlc effects of chloroqume, the abdomen of the pregnant rats was opened carefully The uterine horns were isolated to count corpus leutem, hve fetuses, resorption and still births The fetuses and placentae were removed and washed in cold 1 N sahne Each placenta and fetus was weighed individually and samples were stored in 70% ethanol The samples were &vlded for microscopic, skeletal and shcmg studies and examination The fetuses w~th identifiable abnormaht~es were further processed for skeletal tests w~th alazarm red along with their corresponding controls The details of the procedures have been descrthed previously (13,16)

Study of Teratogentc Effects of Ethanol

Effects of prolonged ethanol consumption by pregnant rats for 15 days lmmedmtely after conception were studied by macro- scoplc and gross anatomical exarmnatlon in the ethanol-treated and corresponding sucrose-fed groups Comparative studies of teratogemc effects of chloroqulne with those of ethanol were also conducted

Methods of Btochemtcal Analysts

The biochemical consequences of ethanol intake by pregnant

FIG 1 Growth retardation produced by chloroqulne admmlstrat~on dunng gestation The two top p~ctures show fetuses from the chloroqume-treated groups The bottom picture, for comparison, is of the fetus from the control group

rats on the developing fetus were examined by studying the incorporation of &fferent radlolSotoplc precursors into their sub- strates in both the liver and brain, and by measuring the steady- state concentrations of different metabolltes

(U-14C)alanlne mcorporatton mto hver glycogen m vtvo The incorporation of intravenously injected (U-14C)alanine Into fetal hepatic glycogen was followed to study the effect of ethanol on hepatic gluconeogenesls A solution of radioactive alanlne (20 mmol) in 50 txl of 0 9% NaC1 (adjusted to pH 6 0), containing 0 5 mCi of (U-14C)alanme, was injected through the tall vein of the pregnant rat After 15 min the ammals were killed by cervical dislocation and fetal livers were frozen In SltU by Wollenberger clamps frozen in liquid nitrogen Glycogen isolation and glucose determination were made as described previously [cf (13)]

Determmatton of protein synthests Protein synthesis was determined in VlVO by measuring the incorporation of a flooding dose of (14C)vallne as described previously (10) On the other hand, m vitro protein synthesis was determined by incubating, the ribosomal and pH 5 enzyme fraction from either hver or brain ussue with (~4C)leuclne The details of protein synthesis determination have been described previously (7) Total protein was determined colonmetncally with bovine serum albumin as protein standard (4)

Determmatlon of m vtvo RNA synthests m fetal bram The effects of prolonged maternal ethanol consumption on the fetal cerebral RNA was determined by determining RNA synthesis in the brains of fetuses from both the ethanol-treated and control

TOXICOLOGY OF CHLOROQUINE AND ETHANOL 79

TABLE 2

LOW INCIDENCE TERATOGENIC EFFECTS OF CHLOROQUINE

Types of Abnormalities Treatment Abnormalities % of Control

Chloroqume (700 mg/kg) for 15 days

Wrist drop 3 5 (79) Clubbed foot N D (76) Broad snout 3 9 (76) Hydrocephaly 2 5 (76) Syndactyly 2 7 (79) Cleft palate N D (29) Brain hqmfication 6 8 (43) Other cerebral and 3 2 (29)

brain defects Wrist joint 4 8 (36) Ankle joint N D (47)

Effects of prolonged chloroqume administration dunng gestation have been studied on the developing fetus compared to the corresponding fetuses from the saline-treated group Low incidences of teratogenlc effects were observed by using macroscopic, slicing and skeletal studms as described in the Method section The results are expressed as percent of control with the number of observations in parentheses N D denotes not detectable

groups (U-3H)urldlne (25 mCl) was injected intravenously through the tail vein. The detmls of (5-3H) into RNA and extraction of radioactive RNA have been descnbed prewously (10)

Determination of tn vlvo DNA synthests tn fetal bram Radio- active thyrmdlne (18 4 Cffmmol) was injected intravenously through the tml-vem. Incorporation into DNA was determined by extracting DNA in 1 6 N perchlonc acid The details of DNA extraction have been described elsewhere (10) Incorporatmn ume course was done at 30-rmn intervals The results are expressed as dpm/txg of DNA at 30 mln after the mjectmn Fetal brain DNA and RNA contents were determined as described prewously (10)

RESULTS AND DISCUSSION

The observed teratologlcal and toxic effects of chloroqume administration to pregnant rats can be divided into two categones 1) teratologlcal effects observed frequently and 2) effects that were observed with lesser frequency. The overall frequency of terato- logical effects of chloroqume were recorded on the basis of all four examination techniques

Frequently Observed Teratologtcal Effects of Chloroqume

Macroscopic studies showed that the adrmmstratlon of chloroqulne to pregnant rats resulted in a slgmficant reduction in the total body weights of the fetuses and of the placentae A generalized

FIG 2 Skeletal abnormalities caused by chloroqume admlnlstratmn dunng pregnancy The picture shows the comparison between normal fetus on the left and chloroqume-treated fetuses showing missing nbs in the center, and bilateral wavy ribs in the picture on the right

80 S H A R M A A N D R A W A T

T A B L E 3

TERATOGENIC EFFECTS OF ETHANOL

Types of Abnormalmes Treatment Abnormalities % of Control

Ethanol-Fed Ankle-joint 6 8 (20) (blood alcohol) Cleft palate 11 1 (20) 190 mg% Growth retardauon 69 8 (20)

Hydrocephaly 3 5 (20) Hydronephrosls 3 1 (20) Syndactyly 6 1 (20)

Effects of prolonged ethanol administration during gestanon have been studied on the developing fetus compared to the corresponding fetuses from the sahne-treated group The details of techmques for the study of teratogemc effects have been described m the Method section The results are expressed as percent of control with the number of observations m parentheses

growth retardat ion o f the fetus was also observed in the chloroqulne- t reated group (Fig 1) compared to the cor responding controls As shown in Table 1, the body size o f the fe tuses f rom the chloroqulne- t rea ted group was decreased These observat ions sugges t that materna l ly admmts te red chloroqulne does cross the feto-placental barrier and exerts t e ra togemc effects on the developing fetus The growth retardat ion at the gestat lonal period o f up to 15 days IS not surpr is ing s ince dur ing the early deve lopmen t the fetus is part tcularly suscept ible to the toxtc effects o f the drugs

Shc lng studies showed that chloroqulne admlnts t ra t lon resul ted m hepa tomega ly in the fe tuses (Table 1) compared to the corresponding controls A h~gh incidence o f hqmf ica t lon o f visceral organs was also observed compared to the controls (Table 1) The s tudy showed that the vtsceral organs , i rrespective o f the locatton in the visceral , cavt ty were affected In certain cases a gene rahzed decay o f the pe r i toneum was also observed

Skeletal examlna t tons showed that the fe tuses f rom the chloroqulne- t reated group showed wavy ribs (Table 1 and Fig 2), part icularly in thir teenth, four teenth and f if teenth rths The other

T A B L E 4

EFFECTS OF MATERNAL ETHANOL CONSUMPTION ON IN VIVO AND IN VITRO FETAL HEPATIC GLUCONEOGENESIS

(~4C)Alanme Incorporation Into Treatment Hepatic Glycogen (ixmoles/g hver)

In Vlvo Study Ethanol-fed group 1 63 ± 0 20 (6) Pair-fed controls 1 70 ± 0 18 (6)

In Vitro Study Sahne I 10 ± 0 18 (6) Ethanol (10 mM) 1 05 ± 0 17 (6) Pyrazole (2 mM) 1 09 ± 0 17 (6) Pyrazole (2 mM) + 1 06 ± 0 17 (6)

Ethanol (10 mM)

N S

N S

N S

Effects of prolonged ethanol administration dunng gestation have been studied on the hepatic gluconeogenesls both in vwo and in vitro m the developing fetuses The detads of techmques for the study of gluconeogenesis have been descrthed in the Method secnon The ad&nons to the Incubation medium m the m vitro studies are hsted in the table The results are expressed ± SEM w~th the number of observations in parentheses

T A B L E 5

EFFECTS OF MATERNAL ETHANOL CONSUMPTION ON IN VIVO AND IN VITRO FETAL HEPATIC AND CEREBRAL PROTEIN SYNTHESIS

Isotope Incorporation Treatment Tissue (cpm/mg protem)

In V1vo Study Control-fetus Liver 2,660 + 300 (6) Ethanol-fetus Lwer 1,587 ± 198 (6)

Control-fetus Brain 140 0 +- 8 0 (6) Ethanol-fetus Brain 112 0 -+ 7 0 (6)

In Vitro Study (pmoles/mln/mg pH 5 × ribosomal protein)

Control-fetus Liver 108 0 _+ 6 0 (12) Ethanol-fetus Liver 68 0 ± 6 0 (12)

Control-fetus Brain 5 76 ± 0 8 (8) Ethanol-fetus Brain 3 9 + 0 7 (8)

Effects of prolonged ethanol administration dunng gestation have been studied on hepauc and cerebral protein synthesis both in vlvo and m vitro in the developing fetuses The details of techmques for the study of protein synthesis have been descnbed m the Method section The additions to the incubation medmm m the m vitro stu&es are hsted in the table The results are expressed ± SEM with the number of observations in parentheses

skeletal abnormal i ty observed upon ch loroqume admlmst ra t lon was that m the fe tuses f rom thts group ribs were f requent ly m l s s m g One o f the h ighes t f requencies o f skeletal abnormaht l e s observed In the chloroqulne- t reated group was the nonoss i f lca t lon o f upper and lower digits o f the fe tuses (Table 1) The skeletal abnormal i t ies observed in the present s tudy sugges t ch lo roqume possibil i t ies o f lack of osstf icat ion process and bone format ion and a poss ible eros ion o f the newly fo rmed bone Irrespective o f whtch is the d o m m a n t m e c h a m s m , tt ts clear that ch lo roqume has a very

T A B L E 6

EFFECTS OF MATERNAL ETHANOL CONSUMPTION ON IN VIVO FETAL CEREBRAL RNA, DNA SYNTHESIS AND BRAIN CONSTITUENTS

Treatment Brain Parameters Control-Fetus Ethanol-Fetus

RNA Synthesis 47,000 __ 5,00 (8) 25,000 _+ 3000 (8) (5-3H) Undlne into p<0 001 polysomal RNA 10 -3 cpm/mg protem)

DNASynthesis 2 8 +- 0 2 0 (8) 1 6 -- 0 18 (8) (14C)thymldlne p<0 01 dprn/tzg DNA/30 mm

Brain Constituents (mg/kg brain wt ) TotalRNA 350 -- 3 3 (10) 270 -- 2 0 ( 1 0 ) TotalDNA 282 -- 2 0 ( 1 0 ) 149 ± 1 5 (10) (RNA/DNA) 1 25 1 88

Effects of prolonged ethanol administration dunng gestation have been studied on cerebral protein synthesis in VlVO in the developing fetuses The detads of techmques for the measurements of brain constitutes and RNA and DNA synthesis have been described in the Method section The results are expressed ± SEM with the number of observations m parentheses

TOXICOLOGY OF CHLOROQUINE AND ETHANOL 81

FIG 3 Wrist drop caused by chloroqume admlmstratlon dunng pregnancy chloroqume-treated fetuses showing wrist drop m (b)

Lz The picture shows the comparison between normal fetus in (a) and

pronounced effect on ossification (up to 100%) process

Less Frequently Observed Teratogemc Effects of Chloroqume

Admlmstratxon of chloroqume dunng pregnancy also resulted m several less frequently observed teratogenlc effects m the fetus These included wrist drop, broad snout hydrocephaly and syndactyly (Table 2) Furthermore, varymg degrees of hqmficatlon of brain were also observed (Table 2) The brain hqmficatlon ranged from seml-llqmd state of the brain to incidences where brain matter was completely replaced by mdky white hqmd Chloro- qume administration also resulted m other defects of ankle, wrist joints, and wrist drops (Table 2 and Fig 3) Marked reduction m body weights 2) and placental weights and shapes have been reported from other laboratories (2)

Teratogemc Effects of Ethanol

As shown in Table 3, a slgmflcant degree of growth retardation was observed m the fetuses from ethanol-fed group and the body weight and body size of these fetuses was lower than the corresponding controls Additionally, ethanol also resulted m cleft palate hydrocephaly, hydronephrosas and syndactyly (Table 3) The teratogemc effects of ethanol and chloroqume showed sxml- lanUes w~th respect to generalized growth retardation and syndactyly (Tables 1 and 3 and Fig 1) The hydrocephaly and hydronephrosls produced by ethanol was not observed m cases of chloroqume administration Furthermore, ethanol administration

also resulted m more pronounced defects of cleft palate compared to chloroqulne The teratogenlc effects of ethanol have been previously reported by this and other laboratories (6)

Blochemwal Effects of Ethanol

Ethanol intake dunng gestation resulted in a number of derangements in the biochemical and metabolic pathways In the fetal liver and brain as compared to the corresponding control group

Effect on Fetal Hepatw Gluconeogenests

In the adult mammahan systems (9,10), and m the newborns suckhng on ethanol-fed dams, it has been observed that ethanol results in an inhibition of hepatic gluconeogenesls-genesls [cf (13)] In fact, at has been suggested that the inhibition of hepatic gluconeogenesas may be the contributing factor in the hypoglyce- mia of ethanol However, in the fetus where the rates of gluconeogenesls are neghglble or very low due to the lack of development of a full compliment of hepatac gluconeogenlc enzymes, ethanol may have no or little effect As shown m Table 4, effects of maternal ethanol consumption dunng pregnancy were investigated on fetal hepatic gluconeogenesls both in VlVO and m vatro As predicted, maternal ethanol consumptton dad not result In a slgmficant change m the lncorporataon of (14C)alamne into hepatic glycogen (Table 4) The addition of pyrazole to the lncubatxon

82 SHARMA AND RAWAT

medium did not slgmficantly alter the incorporation of alanlne (Table 4)

Effects on Fetal Hepatic and Cerebral Protem Synthesis

Protein synthesis studies with the flooding dose of (~4C)vallne showed that both the fetal livers and brains from the ethanol-fed mothers showed a significant inhibition m the m VlVO protein synthesis (Table 5) An inhibition of in vitro protein both m liver and brain of fetuses from the ethanol-fed group was also observed using (~4C)alanme compared to the corresponding controls (Table 5) The inhibitory effect of maternal ethanol consumption dunng gestation and lactation was first reported m early 1975 (7) on the brain protein synthesis It has been subsequently confirmed by other laboratories [cf (10)] Further studies from this laboratory have shown that at least in the adult and newborn brains the inhibitory effect of ethanol on the brain protein synthesis is a &rect effect of ethanol and is independent of the hypothermia generated by ethanol (10)

Effects on Cerebral RNA and DNA Synthesis and Contents

As shown m Table 6, maternal ethanol consumption resulted an slgmficant inhibition m the incorporation of (5-3H)urIdlne into fetal cerebral RNA An mhthItIOn of fetal cerebral DNA synthesis was also observed with the incorporation of (14C)thymIdlne into cerebral DNA (Table 6) Total contents of both cerebral RNA and DNA showed a decrease in the fetal brains from ethanol-fed group compared to the corresponding controls As far as these authors are aware, an inhibitory effect of ethanol on the fetal cerebral

DNA synthesis has been observed for the first time This laboratory has previously reported inhibition of both RNA and DNA synthesis in the brains of newborns suckling on the ethanol-fed dams (10) The RNA/DNA ratio in the fetal brain increased from 1 25 in the control group to 1 88 in the ethanol-fed group (Table 6) Similar effects of ethanol on the brain RNA/DNA ratio have been observed m the newborns suckling on the ethanol-fed mothers (10) Since the brain is especially sensitive to exogenous insults during development, decreased brain weights, smaller s~ze of the brains of the fetuses from ethanol-fed dams (7-9), may be due to the inhibitory effects of ethanol on the synthesis of these macromolecules It IS also possible that the observed behavioral disorders, decreased intelligence quotient (I Q ) and mental retardation, observed m Fetal Alcohol Syndrome may be attributable to the alteration in the metabolic pathways of these macromolecules m the brain We have previously reported that ethanol per se is capable of contributing to these effects even in the absence of its hypothermlc effect

In conclusion, chloroquane and ethanol exert several teratogenlc effects on the developing fetus which are similar, including the generahzed growth retardation of the fetus The hydrocephaly and hydronephrosxs caused by ethanol are not shared by chloroquine The prenatal growth retardation, lowered brain size and brain weights in the fetuses from the ethanol-fed dams could be attributed to the inhibitory effects of ethanol on fetal brain proteins, RNA and DNA It is possible that some of the &fficult~es of behavioral abnormalities, lowered I Q and mental retardation observed In Fetal Alcohol Syndrome could be accounted for by the changes m the metabolic pathways of these important macromolecules in the brain

REFERENCES

1 Colhns, E , Turner, G Six children affected by fetal alcohol syndrome Med J Aust 2606-608, 1978

2 Hopklnson, L , Jackson, F L Effects of chloroqum on growth and metabohsm Nature 202 27-31, 1964

3 Lemome, P , Harousseau, H , Borteyru, J P , Menuet, J C Les enfant de parents alcohohques abnormahes observees A propas de 127 cas Quest Med 25 477-482, 1968

4 Lowry, O H , Rosebrough, N J , Farr, A H , Randall, R J J Biol Chem 193 265-275, 1951

5 Mlrkm, B L , Smgh, S In Mdkm, B L , ed Pennatal pharmacology and therapeutics New York Academic Press, 1976 1-69

6 Randall, C L , Taylor, W J Prenatal ethanol exposure m mice Teratogemc effects Teratology 19 305-312, 1979

7 Rawat, A K Ribosomal protein synthesis m the fetal and neonatal rat bram as influenced by maternal ethanol consumption Res Commun Chem Pathol Pharmacol 12 723-732, 1975

8 Rawat, A K Psychotroplc drug metabohsm m fetal alcohol syndrome Adv Exp Med Blol 132561-568, 1980

9 Rawat, A K Pharmacological and toxicological considerations in

fetal alcohol syndrome In Messlha, F , Tyner, J , ed Alcohohsm, A perspecnve New York PJD Pubhcanons, 1980 149-176

10 Rawat, A K Nucleic acid and protem synthesis mhlbltlOn m the developing brain by ethanol m the absence of hypothermla Neurobe- hav Toxlcol Teratol 7 161-166, 1985

11 Robinson, G C Heanng loss and other defects m infants treated with qulnme Pc&ames 32 150-152, 1963

12 Schar&an, J In Schr&an, J , ed Drugs as teratogens New York C R C Press, 1978

13 Sharma, A , Rawat, A K Teratogenlc effects of hthmm and ethanol m the developing fetus Alcohol 3 101-106, 1986

14 Sulhvan, G E , Takacs, E Comparanve teratogemclty of py- rlmethamlne in rats and hamsters Teratology 4 210-216, 1972

15 Tanlmura, T , Lee, S Discussions on the teratogenlclty of quinine to humans Teratology 6 122-124, 1972

16 Wdson, J G , Warkany, J In Wdson, J , Warkany, J , eds Teratology Pnnclples and techmques Chicago Umvers~ty of Chi- cago Press, 1965

toxicological consequences of chloroquine and ethanol on the developing fetus

Documents