science
Chemicals affect brain in diverse ways FASEB participants hear about learning transfer, changes in synthesis of protein and RNA, effects of mercury and zinc "The experiments," one scientist said of his work, "were all done in a scientific looking laboratory." With visions of bubbling kettles, blinking lights, and hunchbacked assistants, the listener sits rapt, wondering if the Federation of American Societies for Experimental Biology has delivered over its annual meeting to alchemists, charlatans, swamis, or worse. It has not. (The quote above was from a perfectly respectable paper detailing experiments on the "influence of expectation, setting, and previous drug experience" on the marijuana-induced "high." The effect, incidentally, was found to be great.) What FASEB has done has been to bear witness to the growing thrust of scientific studies into the complex relationship of mind and body and—most strikingly— into the chemical events at the cellular level that may explain bodily functions and behavior. Learning. Dr. Georges Ungar and his Baylor University team traveled to Chicago to recount continuing progress in their work on chemical transference of learned behavior. Using fish, Dr. Ungar has shown that color preference can apparently be relayed from subjects taught to avoid one side—the green-lighted side, for example—of the tank. Dr. Ungar used an electric shock to cause the fish to avoid one side or the other of redblue, blue-green, and yellow-orange lighted tanks. Extracts were then prepared from the brains of the trained fish and injected into the cranial cavity of recipient fish which then, as a group, tended to perform as the trained specimens had been taught. Dr. Ungar and his colleagues now plan a massive, 40,000-fish experiment, training half the fish for blue and half for green avoidance. This group, Dr. Ungar hopes, will provide sufficient
brain material to attempt an identification of the peptide code words that he believes are the keys to learned behavior. By identifying the peptides corresponding to avoidance of the different colors, "we hope to obtain extremely valuable information of the code words," Dr. Ungar says. Isoprinosine. Participants at the FASEB meeting also heard that isoprinosine—an inosine alkylaminoalcohol complex—has been approved for general use as an antiviral agent in humans by the Argentine government. The drug, coded NPT-10381 by its manufacturer, Newport Pharmaceuticals International, Newport Beach, Calif., has received previous attention for its "memory enhancing" properties. Dr. Paul Gordon of the Chicago medical school, told FASEB that the drug "has its origin in the field of biopsychology rather than virology," but whatever its origin, isoprinosine represents an intriguing story of the interplay of viral infection and the incorporation of RNA and protein into brain cells. "Isoprinosine exerts very important biochemical and functional actions in uninfected tissues," Dr. Gordon asserts. And he adds that both memory deposition in animals and viral replication depend upon similar macromolecular syntheses. In tests in rats, isoprinosine, given at levels of 300 mg. per kg. per day in drinking water for three days, in-
creases the number of polyribosomes in brain cells of aged—two-year-old— animals, Dr. Gordon notes. Aged animals without drug treatment show a decided reduction in absolute number of brain polyribosomes in comparison with both young normal rats and drugtreated animals. In like manner, Dr. Gordon has shown that isoprinosine enhances the rate of synthesis of cytoplasmic RNA, which he presumes is messenger RNA. In these tests the Chicago investigator used tritiated orotic acid to produce labeled RNA and calculated specific activities of the labeled RNA from acid-insoluble counts per unit RNA in the cytoplasm. Viruses. Dr. Gordon and his coworkers, Dr. E. R. Brown and Dr. B. Ronsen, also examined monkey kidney cells in tissue culture for isoprinosine's effect on both RNA and protein synthesis. In these tests viral infection was an additional condition, and it is these experiments which indicate that isoprinosine acts both to offset the effects of aging and to combat rapid-acting viral infections. "In uninfected monkey kidney cells," Dr. Gordon told FASEB scientists, "NPT-10381 synchronously enhances the incorporation of both rapidly labeled RNA and radioactive amino acids into polyribosomes." Polyribosomal RNA was increased in specific activity by 25 to 250%, depending on the polyribosome size category, Dr.
Drug halts viral growth in tissue cultures Virus titer1
Treatment
Day 0
Polio type 3
Control NPT-10381
1.3 X 105 1.3 X 105
1.0 X 107 1.5 X 104
Adenovirus type 10
Control NPT-10381
1.5 X 103 1.5 X 103
1.2 X 104-5 1.0 X 103
Influenza a2 Hong Kong
Control NPT-10381
1.2 X 102 1.2 X 102
1.1 X 1 0 4 1.0 X10 2 -*
Influenza Pr-8 strain
Control NPT-10381
1.5 X 103 1.5 X 103
1.0 X 104-5 1.0 X 103-5
Herpes Lu strain
Control NPT-10381
1.5 X 103 1.5 X 103
1.0 X 104-5 1.0X10 3
Virus
Day 3
a Virus particles per milliliter inoculum. APRIL 26, 1971 C&EN 27
Gordon says. Incorporation of radioactive amino acids—an event reflecting the protein synthesis rate—increased to a similar degree. In the presence of the Pr-8 strain of influenza virus (see table), isoprinosine was found to induce a selective depression of RNA label incorporation into polyribosomes in both the heavy and light categories. Amino acid incorporation was also altered. The drug, in the presence of virus, also decreases the fraction of total messenger RNA label found in polyribosomes by about 30°/o, Dr. Gordon notes, suggesting that the average association of ribosome and messenger RNA is less effective, or at least altered. "These results," Dr. Gordon concludes, "identify a metabolic response to drug which is dependent for its character upon the presence or absence of virus infection." In the tissue culture system with monkey kidney cells, NPT-10381 induces a "striking inhibition in the replication of virus antigen protein," Dr. Gordon observes. Focusing on the two strains of influenza virus, Dr. Gordon emphasizes that there is a drug-induced 1 to 1.5 log difference in virus titer by the third day. Stressing that the interpretation must be tested further, Dr. Gordon offers the view that ribosomes become altered in structure through the action of isoprinosine so as to translate host messenger RNA more effectively and virus messenger RNA less effectively than in controls. Clinical trials in Argentina—where doctors have reported virtual disappearance of flu symptoms 24 hours after drug treatment was begun—seem to underscore Dr. Gordon's contentions. At this point, Newport Pharmaceuticals is testing isoprinosine under an Investigative New Drug clearance from the U.S. Food and Drug Administration. The firm says that the drug has thus far been relatively free of toxic side effects. Argentine investigators report that it has been tolerated by children and aged people as well as other adults. Although isoprinosine's intimate relationship to both RNA synthesis and viral replication might seem to bear directly on recent theories concerning viral-induced cancers, Newport Pharmaceutical is not touting the drug as an anticancer material. Dr. Gordon notes that flu viruses are rapid-acting, whereas cancer's long course would seem to implicate slow-acting viruses that remain viable for extended time periods. Isoprinosine will almost certainly be evaluated for anticancer activity in due course, but if it can indeed help relieve man of flu 28 C&EN APRIL 26, 1971
Dr. Georges Ungar
and cold symptoms, it will merit in every sense the appellation of "wonder drug," whatever its effects on cancer. Just as isoprinosine apparently acts to build up brain RNA and protein, alcohol may act to depress synthesis of these vital macromolecules. Working with mice exposed for two weeks or longer to a 10% alcohol solution, Dr. Sujata Tewari and Dr. Ernest P. Noble of the University of California, Irvine, have discovered a significant decrease in the ability of the test animals to make brain proteins and RNA. Uptake of radioactive leucine was decreased in both the ribosomes and pH-5 enzyme fraction (the soluble fraction of the cell) by chronic alcohol consumption. By first injecting tagged amino acid into "alcoholic" and control mice and then examining tissue samples, the Irvine-based team showed that brain protein of the alcohol-adapted mice had about one half of the radioactivity of the controls two hours after amino acid injection. In vitro experiments indicate that the initial reaction of protein synthesis—the activation of amino acid to form the complex, aminoacyl tRNA—was severely depressed in alcohol-adapted mice. Dr. Tewari and Dr. Noble also showed that incorporation of tritiated orotic acid into RNA was markedly depressed in the pH-5 enzyme fraction in alcoholtreated mice, with ribosomal RNA incorporation also decreased, but to a lesser extent. Mercury. In another development at the FASEB meeting relating to brain damage, Dr. T. W. Clarkson, H. Small, and Dr. Tor Norseth (C&EN, April 19, page 12) described studies with mercury-binding, sulfhydryl-containing resins aimed at reducing absorption of methylmercury compounds from food and increasing the rate of ex-
cretion from the body. Methylmercury is the extremely toxic form of the metal, which has been responsible for several dozen fatalities and hundreds of cases of brain damage. Excretion of methylmercury, Dr. Clarkson says, occurs mainly through the feces. Intravenous injection of 175 micrograms of labeled mercury (as methylmercury chloride) into rats showed 20 micrograms excreted in the bile the first day, but only 4 micrograms appeared in the feces. Since some of the fecal mercury may have come from sources other than bile, Dr. Clarkson calculates that probably 90% or more of the mercury excreted in the bile is reabsorbed. Of various metal-binding resins tested, sulfhydryl types were most successful at binding mercury and preventing reabsorption. Tested against controls (where controls and test animals received a single injection of radioactive methylmercury), mice given 1% by weight resin in their food excreted mercury about two and a half times as rapidly. Upon sacrifice (after 12 days), the resin-treated animals showed levels of mercury in the brain, liver, and kidneys that were reduced by a factor of five when compared with controls. Mercury was undetectable in the blood of treated animals. To test the ability of resin to reduce absorption of methylmercury from food, Dr. Clarkson's group fed mice food containing radioactive methylmercury; the experimental group also received 1% by weight sulfhydryl resin in its food. The uptake of methylmercury was followed by whole body counting. The control group, Dr. Clarkson says, accumulated radioactivity much more rapidly than did resin-treated animals; after 12 days, radioactivity in controls was four times as great. In part, Dr. Clarkson adds, this may be a result of elevated excretion in resin-treated animals, but he estimates that a 50% reduction in uptake was also achieved. Zinc. Zinc, another metal in the mercury group, was also the subject of several papers at the Chicago meeting. Norethindrone and mestranol—two contraceptive hormones —both act to lower plasma zinc concentration in rats, investigators at Veterans Administration Hospital, Washington, D.C., told the meeting. Mestranol, which has been implicated in many of the side effects in women taking oral contraceptives, according to VA's Louis D. McBean, Dr. J. Cecil Smith, and Dr. James A. Halsted, caused the most significant changes in zinc levels. Pregnant, zinc-deprived rats receiving contraceptive hormones
have experienced increased fetal mortality, whereas a diet containing sufficient zinc (50 p.p.m.) did not affect fetal survival. Thus, an increased zinc requirement for metabolism of excess hormone could explain the lowered zinc plasma levels. Zinc may also play a role in schizophrenia, scientists Carl C. Pfeiffer, Venelin Iliev, and J. Cawley at the New Jersey Neuropsychiatric Institute, Princeton, N.J., told FASEB. Their data indicate that, with zinc and manganese dietary deficiencies, the schizophrenics' tissues may accumulate copper and iron, causing malfunction of enzymes. The altered enzymes may, in turn, cause overstimulation of the brain.
ical school first isolated metallothionein from horse kidney in 1960 and later from human kidney. The Harvard workers showed that metallothionein binds cadmium, zinc, copper, mercury, and other trace elements with sulfhydryl groups from the amino acid cysteine, which constitutes 20 to 25°/o of metallothionein. They suggested that metallothionein may play a vital role in the storage and detoxification of trace elements such as cadmium, zinc, and copper. With the aid of radioactive tracer studies, Dr. Evans and his coworkers have shown that a large fraction of copper in both the intestine and liver is bound to metallothionein. In the intestine, copper is bound to metallothionein before absorption into the blood. Then copper is transported by Metallothionein, a metal-binding pro- albumin in the blood to the liver tein, may play an important role in where the metal is bound to metalloWilson's disease, Dr. Gary W. Evans thionein for storage, Dr. Evans says. of the human nutrition research diDr. Evans and his associates have vision, U.S. Department of Agricul- isolated metallothionein from the liver ture, Beltsville, Md., told a nutrition of a patient with Wilson's disease and session at the Federation of American from a control patient. Copper analSocieties for Experimental Biology ysis showed that metallothionein meeting in Chicago. Working in col- from the Wilson's disease patient conlaboration with Dr. W. E. Cornatzer of tained a much higher level of copper. the University of North Dakota med- Further studies using equilibrium diical school, Grand Forks, and Dr. Reu- alysis indicated that the protein from ben S. Dubois and Dr. K. Michael the patient with Wilson's disease had Hambidge of the University of Colo- a higher affinity for copper than did rado medical center, Denver, Dr. the protein from the control patient. Evans also finds that metallothionein In the liver of a normal patient, exis involved in copper absorption in cess copper is excreted into the bile the intestine as well as copper stor- and ultimately eliminated in the feces. age in the liver. In addition, copper is released from Defect. In Wilson's disease, the liver to the blood tightly bound to metallothionein is apparently altered, a globular protein, ceruloplasmin, and the protein has a higher than which functions as a circulating resernormal affinity for binding copper, the voir for copper. scientists believe. Only four or five Affinity. However, these mechapeople in a million have Wilson's dis- nisms are impaired in the patient with ease, an inherited defect in copper Wilson's disease. As a result of a metabolism that causes toxic copper genetic error, metallothionein in the levels to deposit in the liver and Wilson's disease patient has a higher brain. Signs of the disease are a affinity for copper. Furthermore, Dr. greenish, brownish ring that shows up Evans and his associates think that in the eye, tremors, incoordination, ceruloplasmin has decreased ability to and any manifestation of liver disor- bind copper. der. The symptoms usually appear As the disease progresses, copper in patients during adolescence. Cop- accumulates in the liver, and it evenper concentrations in the liver of dis- tually exceeds the storage capacity of eased patients range from 100 to 1000 that organ. The excess copper is micrograms per gram of wet weight, spilled over into the blood. Since it compared to 5 micrograms per gram is not incorporated into ceruloof wet weight in normal patients, ac- plasmin, the copper is deposited in cording to Dr. Evans. Scientists have other organs such as the brain. tried to explain the genetic abnormalThese experiments may help resolve ity that produces the disease since Dr. the controversy regarding the fundaKinnier Wilson first described the mental cause of Wilson's disease, Dr. syndrome in 1910. Evans says. The work may aid cliniDr. Bert L. Vallee and his as- cians in prescribing more effective sociates at Harvard University med- treatment for the disorder.
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Protein is defective in Wilson's disease
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APRIL 26, 1971 C&EN
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