Environmental Health Perspective8

Lead Aggravates Viral Disease and

Represses the Antiviral Activity

of Interferon Inducers

by Joseph H. Gainer*Lead acetate was administered continuously in the drinking water to CD-1 male mice

beginning at 4 weeks of age. An LD,o02o of the lytic viruses or 300 plaque-forming unitsof RLV was inoculated intrapertioneally at 6 weeks of age. Lead increased the responseof the mice to all classes of viruses against which it was tested: an RNA picornavirus-encephalomyocarditis (EMCV), a DNA herpesvirus-pseudoribies, an RNA leukemia-virus-Rauscher leukemia (RLV), an RNA arbovirus B-St. Louis encephalitis, and an

RNA arbovirus A-western encephalitis. Most studies were performed between lead andEMCV. Increases in EMCV mortality in lead treated mice over controls ranged from2X at a lead level of 0.004M to 7 x (100% mortality) at a 0.1M lead level. Splenomegalywith spleens 800 to 1100 mg in weight containing high titers of RLV occurred in lead(0.03M)-treated mice 3 and 6 weeks after RLV inoculation; spleens or RLV controlswere normal in weight (200 mg) and were free of virus. Lead did not reduce the pro-

tective effect of mouse interferon (IF) against the lethal action of EMCV, but it didrepress the EMCV antiviral effect of poly I/poly C (PIC) and of Newcastle diseasevirus (NDV) against EMCV mortality. These data indicate several new facts concern-

ing adverse effects lead may have on an animal: (1) lead aggravates viral disease,most likely in part, through reduced IF synthesis; (2) lead represses the anti-EMCVprotective effects of both PIC and of NDV, which, in other reports, were shown to in-duce IF in radioresistant macrophages (PIC) or in radiosensitive lymphocytes (NDV);(3) lead may then be said to repress IF induction in two kinds of cells; (4) however,lead does not inhibit IF action.

IntroductionOne of the research programs of the Na-

tional Institute of Environmental HealthSciences was to screen various classes ofchemicals for their adverse or beneficial ef-fects on experimental viral infections. Stud-ies on interactions of this nature appearwarranted for several reasons. (a) A direct

*National Institutes of Environmental Health Sci-ences, Research Triangle Park, North Carolina 27709.

Present address: Bureau of Veterinary Medicine,FDA, 7B-45, Parklawn Building, Rockville, Mary-land 20852.

consideration of this research may lead tothe fact that elevated environmental chemi-cal levels do in fact aggravate or they mayalleviate viral infections. (b) It is knownthat cigarette smoking aggravates influen-zal infection in man (1), and many chemi-cals have been shown to increase viral in-fections experimentally (2). (c) Insofar aslead poisoning and viral infection is con-cerned, possible synergism between leadpoisoning and eastern encephalitis viral(EEV) infection was observed "naturally"in a dead calf in Florida wherein EEV was

May 1974 113

isolated from the brain, the calf had a viraltype of encephalitis histologically, and 20ppm lead was present in the kidney of thecalf. (J. H. Gainer, unpublished observa-tions, Fla. Dept. Agriculture, Kissimmee,Fla, 1964). (d) A viral infection, being a dy-namic (and not a static) force within a cellor animal, presents problems diverse froma strictly classical toxicological sense-thehost must cope with the background chemi-cal poisoning and then confront itself withthe virus that exerts very powerful forceson the basic biochemical machinery of thecell. In contrast, then, to the simple straight-forward toxicological approach with onechemical-one host response, here we havethe chemical, the host, and the virus, andthe virus itself exerts many pressures thatjust do not exist in the single chemicaltoxicological study. In addition the chemicalmay influence viral absorption and replica-tion directly and subsequently may influencethe host's defense mechanisms against thevirus.

Metallic salts represent a diverse groupof naturally occurring biologically activecompounds; and certain toxic metals, aswell as essential metals, perhaps, are beingmobilized by man's activities and thereforeare being increased in the immediate en-vironment near to man. Lead has beenincriminated as the metal that is being mobi-lized to the greatest extent by man (3). Metal-lic salts appeared then to be a logical groupof compounds that should be examined fortheir interactions with viral disease.The intact mouse rather than tissue cul-

tures was chosen as the test host for inter-actions between metals and viruses for cer-tain reasons. Although interactions betweenmetals and viruses most certainly will occurin tissue cultures, it is almost impossible toextrapolate tissue culture data to wholeanimals and to man because of the manydifferences between a highly artificial and;relatively simple tissue culture system andthe complex whole animal. The animal hasselective absorptive and excretory mecha-nisms for the chemical that the tissue cul-ture system most likely lacks. The animal

may concentrate the metal in a specific tis-sue for which the virus has a special affinity,cobalt and the encephalomyocarditis virus(EMCV) both affecting the heart, for ex-ample (4). This argument may post disad-vantages to certain studies, however; forexample, cadmium, which concentrates inthe kidney, should ideally be interacted witha virus that infects the kidney and not onethat infects the heart or brain, as has beendone (5). Certain metals, because of theirlack of adequate solubility in tissue culturemedia (lead, for example), present anotherbarrier to the successful conduct of tissueculture studies. The mouse is relatively in-expensive, and it therefore can be used insufficient numbers to permit the acquisitionof sufficient data for statistical analysis.The mouse has been well characterized forinfection studies with several classes of vi-ruses. It has been argued that tissue culturesrather than whole animals should be used,because mechanisms are more easily de-finable in tissue cultures; this may be thecase,. but data that follow illustrate thatmuch in terms of mechanisms has beenlearned in the intact animal.

This paper describes in mice (a) the in-teractions between lead treatment and fivedifferent viral infections, (b) interactionsbetween lead treatment and interferon ac-tion, and (c) interactions between lead treat-ment and the antiviral actions of the inter-feron inducers poly I/poly C and Newcastledisease virus.

MethodsMetals, Mice, Viruses

A screening program was begun in whichthe salts of metals were interacted with afew viruses in CD-1 male mice (CharlesRiver Breeding Farms, Wilmington, Mass.).Metals were administered continuously indemineralized drinking water, beginningwith mice at 4 weeks of age. A safe con-centration of a metal was determined as themaximum concentration of the metal themice could tolerate without appreciable re-duction in liquid consumption or appreciable

Environmental Health Perspectives114

loss in weight. Viruses used were commonlaboratory strains; the viruses were inocu-lated intraperitoneally (IP) at 6 weeks ofage. Death rate was the parameter used inthe lytic viral infections; splenomegaly wasfollowed in the Rauscher viral infections.Lead acetate was found to be the least toxicof the metallic salts tested; however, leadwas found to result in a large magnitudeof synergism of the viral disease response.In fact, of several metals tested, NaAsO2,CdSO4, CoS04, HgCl2, NiSO4, and lead ace-tate, lead acetate was the most potent "syner-gizer" of viral infections (4-6).

Interferon Studies

To attempt to delinate modes of action ofthe lead-enhanced virus-induced disease, in-terferon (IF) was considered to be involvedin that IF is one of the first lines of de-fense an animal has against a viral infec-tion (7). Virus titers and IF levels wereexamined sequentially in brain and spleenof mice infected with the encephalomyo-carditis virus (EMCV). Preliminary assaysof the titers of virus and of IF in spleensand brains of virus-control mice and of lead-treated EMCV-infected mice killed or dying2 to 4 days after viral inoculation revealedhigh titers of virus and of IF' in tissues fromboth groups of mice. Lack of sufficient timeprevented careful study of these two para-meters to determine if in fact the lead-treated, virus-infected mice had lower IFtiters and higher virus titers than did thethe nontreated virus-infected control mice.Nevertheless, lead-treated EMCV infectedmice did contain high titers of EMCV, in-dicating that these mice were in fact dyingof a virus-induced encephalitis.

Earlier studies had shown that arsenicalsrepressed the antiviral activity of the IFinducer poly I/poly C for EMCV mortality(8). Poly I/poly C has been shown to pro-tect mice against EMCV mortality, the me-chanism of action being through the synthe-sis and action of IF (9). Recently it wasreported that poly I/poly C induced IF syn-thesis in large radio-resistant macrophageswhereas Newcastle disease virus (NDV),

which will also induce the synthesis of IF,induced it in radiosensitive lymphocytes(10, 11). The effect of lead acetate on theantiviral activity of poly I/poly C and ofNDV was determined. The method of pro-cedure was simply that mice were laden withthe lead as in the straight virus procedures.Poly I/poly C (Biopolymers, Chagrin Falls,Ohio, lot no. 12) or NDV was administeredIP, and 2 or 24 hr later, respectively, anLD9. of virus was administered. A commeri-cal lot of NDV (La Sota strain, Bi typevaccine, American Scientific Laboratories,Madison, WI) was used. Deaths were tal-lied and differences were compared. The in-terferon inducers protected against EMCVmortality whereas the lead-treated mice werenot protected by either IF inducer fromdying with EMCV infection. Lead was thengiven at various times with regard to thepoly I/poly C to determine certain time se-quences of lead repression of IF synthesis.Lead was also tested against preformedmouse interferon in vitro and in vivo todetermine if it would influence the antiviralactivity of the interferon.

ResultsLead and Viral Disease

Table 1 shows that lead acetate exagger-ated the response of mice to all virusesagainst which it was tested. Although thedifferences between virus controls and leadtreated were not statistically significant inthe case of the St. Louis encephalitis andthe western encephalitis virus, there was ahigher per cent morality in the lead-treatedmice than there was in the controls in allcases. The concentration of lead used withthe pseudorabies virus and with the twoencephalitis viruses was not as high as inthe case of EMCV. Spleens in the lead-treated, Rauscher virus-infected mice werevery large; and high titers of virus werepresent in lead-treated Rauscher virus-infected spleens (up'to 108 PFU/gram); vi-rus was not recovered from virus-controlspleens.

May 1974 115

Table 1. Effect of lead acetate on virus mortality and on Rauscher virus splenomegalyin mice.,

No. dead/no. inoculated and per cent mortalityDemineralized Lead

Concentration of water acetate-Virus lead used, M control mice treated mice P

Encephalomyocarditis 0.01 and 0.004b 0/31, 0% 7/32, 22% <0.050.05 6/41, 15% 9/21, 43% 0.040.10 6/41, 15% 11/22, 86% <0.01

Pseudorabies 0.0003 b 4/34, 12% 6/15, 40% <0.050.003 b 6/34, 12% 4/14, 29% 0.06

St. Louis encephalitis 0.03 b 5/23, 22% 7/20, 35% N.S.Western encephalitis 0.015 and 0.03 b 2/20, 10% 5/39, 13% N.S.

Rauscher leukemia 0.03 204± 31, 3WPI; 858 ± 96, 3WPI 00142±12, 6WPI; 1048±374, 3WPI <0.01

Mice were treated with lead acetate beginning at 4 weeks of age; at 6 weeks they were infected withviruses. Mortality patterns were followed in the first 4 viruses; spleen sizes and splenic virus content wereobserved in the Rauscher leukemia infections.

b Unpublished findings; other data from Gainer (5, 1 6).e Spleen weights in mg with standard deviations; WPI denotes weeks after virus inoculation.

Table 2. Effect of lead acetate on the antiviral activ-ity of poly I/poly C and on Newcastle disease viruson EMCV mortality.,

Virus mortalityDemineralized

Lead waterInterferon inducer acetate controls P

Poly I/poly C 12/19, 63% b 1/20, 5% 0.001Newcastle diseasevirus (NDV) 10/15, 67% * 20/74, 27% 0.008

' Lead acetate was begun at 4 weeks of age. At 6weeks poly I/poly C and NDV were given IP, fol-lowed 2 and 24 hr later, respectively, with virus.Data are from Gainer (12).

b Lead acetate, 0.03M.° Lead acetate, 0.003M.

Lead and Interferon Inducers

The interactions of lead acetate with polyI/poly C and with NDV in their antiviralprotective activities against EMCV morta-lity are presented in Table 2. The lead levelin the poly I/poly C study was tenfoldthat in the NDV study, and the poly I/polyC alone protected against EMCV mortalityto a greater magnitude than did the NDV,it is not possible to make direct comparisonsbetween lead and poly I/poly C and leadand NDV. Chessboard experiments to workout these interactions in intricate detailwere not performed.

Three concentrations of lead acetate, 0.003,0.01, and 0.03M, were interacted with 0.2mg/kg of poly I/poly *C. The 0.03M leadexerted as high a reversal activity againstpoly I/poly C, 9/10, P = 0.01, as did the ten-fold concentration of lead, 0.03M, 7/9, P =

0.05; poly I/poly C control mortality was5/17, 29%. The intermediate lead concentra-tion of 0.O1M resulted in a mortality of7/10, P = 0.07 (12).

Temporal Relationships ofLead and Poly I/Poly C

Table 3 presents the results of temporalrelationships between the administration oflead acetate and poly I/poly C. Lead given

Table 3. Influence of the time of administration oflead on the antiviral activity of poly I/poly C inmice.'

Lead treatment Mortality P

1 hr before poly I/poly C 11/20, 55%o NS4 hr after poly I/poly C 5/16, 31% 0.05

24 hr after poly I/poly C 3/20, 15% 0.01Virus controls, no lead,no poly I/poly C 27/41, 66o -

'Six-week-old mice were used. Lead acetate, 100mg/kg, was given IP at times before and after PolyI/Poly C, 0.3 mg/kg, IP. EMCV was given IP 1 hrafter poly I/poly C. Data are from Gainer (12).

Environmental Health Perspectives116

1 hr before poly I/poly C repressed nearly allof poly I/poly C's antiviral activity. Whengiven 4 hr after poly I/poly C, lead stillreversed the protective effect to some ex-tent; but 24 hr after poly I/poly C, lead didnot have any influence.

Effect of Lead on Interferon Action

Mouse interferon was prepared by inject-ing poly I/poly C, 7.5 mg/kg, IP into 8 weekold C3H male mice (Jackson Labs., Bar Har-bor, Maine). The animals were exsangui-nated 4-5 hr later. Plasma from heparinizedblood was pooled with triturated spleensand lungs, this mixture comprised the IFpreparation. Plasmas, spleens, and lungswere collected from normal untreated miceand served as control preparations. Leadacetate when administered prior to the ad-ministration of IF did not influence theprotective activity of the interferon (Table4). This lack of effect of the lead on IF ac-tion compares favorably with in vitro studiesin which lead at 10- and 10-5 M, when addedto IF preparations, did not inhibit the an-tiviral activity of IF against the vesicularstomatitis virus (12). In the in vitro studiesthe difficulty was encountered that lead isquite insoluble in tissue culture mediums;it is conjectured that, because of insolu-bility of the lead acetate in tissue cultures,it was probably not possible to achieve suf-ficiently high concentrations to test ade-

Table 4. Effect of lead acetate on the antiviral activ-ity of interferon against encephalomyocarditisvirus mortality in mice.'

Treatment Mortality,no. dead/

0 hr 2 hr 4 hr total PLead acetate, Interferon Virus 11/20 0.01100 mg/kgSaline Interferon Virus 11/20 0.01Saline Normal Virus 20/21

mousetissues(controls)

'Five-week-old mice were injected three times eachIP as stated. Mortality was followed. Data are fromGainer (12).

quately the effects of lead on interferonaction.

DiscussionData presented show that lead poisoning

aggravated the response of mice to the fiveviruses against which it was tested. Fourviruses are of the RNA type, and one, thepseudorabies, is a DNA virus. The concen-trations of lead used were not the samewith every virus, so that one is uncertainthat the magnitude of virus stimulationfrom virus to virus is really comparable. Inaddition, the dose of virus used is hardlycomparable from one virus to the next, thenumber of virus particles per infectious unitbeing of considerable consequence as towhether or not a virus disease process maybe exaggerated by a chemical poisoning.Nonetheless, responses to all viruses in themice were exaggerated by the lead treat-ment, although not significantly so in thecase of the two encephalitis viruses. It ispossible that the lead had not reached strongvirus-enhancing levels in the brains, whereit would most likely have significant effectwith the encephalitis viruses. It is suggestedthat the lead is attacking a common bio-chemical parameter in the infectious process(such as interferon), rather than a specificbiochemical pathway related to one partic-ular virus only. Although the determina-tions of virus titers and IF levels were notsatisfactory and did not delineate particularrelationships between the two entities, otherinterrelationships between lead poisoningand IF formation and action did lead tovery significant results. Lead poisoning didnot inhibit interferon action either in vitroor in vivo. This finding would suggest thatfor IF to be active another protein is notformed to account for the antiviral action;this contrasts with earlier findings in whichit is reported that IF results in synthesisof a secondary protein that actually pro-vides for the antiviral action (7). In thecase of lead poisoning, the interferon isfully active in its presence; this suggeststhat if the lead inhibits RNA synthesis,as has been reported (13), it is not in-

May 1974 117

hibiting whatever RNA synthesis is occurr-ing as a primary effect of interferon action.Lead poisoning inhibited the anti-EMCV

protective effect of poly I/poly C, presum-ably in large macrophages, and of Newcastledisease virus, presumably in small lympho-cytes. These findings suggest strongly but donot confirm unequivocally that the mechanismof lead enhancement of viral disease isthrough depressed interferon synthesis. Sincethe lymphocytes are radiosensitive, onemight predict that lead poisoning would addor would synergize with radiation-inducedinjury to further intensify viral disease re-sponses.

Interactions of lead poisoning with Raus-cher virus infection affords other points ofdiscussion in addition to probable effects oflead poisoning on the defense mechanisms.Lead poisoning causes a myriad of patho-logical effects, one of the most noteable ofwhich is the induction of anemia (14)through an inhibition of 8-aminolevulinicacid dehydratase and heme synthetase whichare critical for hemoglobin synthesis. Ebertet al. (15) illustrated recently that in RLVinfection, reduced 8-aminolevulinic acid syn-thetase activity was associated with the de-velopment of anemia and splenomegaly. ThusRauscher virus infection and lead poisoningmay be acting synergistically through in-hibition of similar enzymes involved in hemo-globin synthesis.

Sequential studies on the plasmas andtissues in the lead-poisoned animals aftervirus infection for such parameters as anti-body, leukocyte counts, interferon, and virustiters would clarify several issues concern-ing mechanisms involved in these interac-tions. The temporal relationships found herebetween lead and poly I/poly C indicatesclearly that lead disrupts interferon synthe-sis and not interferon action.

SummaryThe administration of lead acetate in the

drinking water aggravated the response ofmice to five viruses against which it wastested: encephalomyocarditis (EMCV), pseu-dorabies, Rauscher leukemia, St. Louis ence-

phalitis, and western encephalitis. On treat-ment with 0.lM lead, EMCV mortalityincreased sevenfold (to 100%o mortality)and spleens of mice treated with 0.03M leadand infected with the Rauscher leukemiavirus weighed 800-1100 mg and containednearly 10 8 PFU of virus per gram 3 and 6weeks after virus inoculation; Rauschervirus-infected control spleens were normalin weight and did not contain any virus.Lead did not inhibit interferon (IF) ac-

tion in mouse embryo cultures in its inhibi-tion of plaque formation by the vesicularstomatitis virus, and it did not inhibit IFaction in that it did not reduce the anti-EMCV protective effect of IF against EM-CV mortality in the intact mouse. Howeverlead did repress the EMCV antiviral effectof poly I/poly C (PIC) and of Newcastledisease virus (NDV) against EMCV mortal-ity. These data indicate several new factsconcerning adverse effects lead may havewith respect to a viral infection: (1) leadaggravates viral disease, most likely in partthrough reduced IF synthesis, as a result ofreduced RNA synthesis; (2) lead repressesthe anti-EMCV protective effects of bothPIC and of NDV, which, in other reports,were shown to induce IF in radioresistantmacrophages (PIC) and in radiosensitivelymphocytes (NDV); lead may then be saidto have the capacity to repress IF synthesisin at least two kinds of cells; (3) however,lead does not inhibit IF action.

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