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 red-blue, 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 alkylaminoalco-hol 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 drug-treated 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 iso-prinosine'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." Polyribo-somal 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

Polio type 3

Adenovirus type 10

Influenza a2 Hong Kong

Influenza Pr-8 strain

Herpes Lu strain

Treatment

Control NPT-10381

Control NPT-10381

Control NPT-10381

Control NPT-10381

Control NPT-10381

Virus titer1

Day 0 Day 3

1.3 X 105 1.3 X 105

1.5 X 103

1.5 X 103

1.2 X 102

1.2 X 102

1.5 X 103

1.5 X 103

1.5 X 103

1.5 X 103

1.0 X 107

1.5 X 104

1.2 X 104-5

1.0 X 103

1.1 X10 4

1.0 X102-*

1.0 X 104-5

1.0 X 103-5

1.0 X 104-5

1.0X103

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), isoprin-osine 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

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 alcohol-treated 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 mest-ranol—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

28 C&EN APRIL 26, 1971

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.

Protein is defective in Wilson's disease Metallothionein, a metal-binding pro­tein, may play an important role in Wilson's disease, Dr. Gary W. Evans of the human nutrition research di­vision, U.S. Department of Agricul­ture, Beltsville, Md., told a nutrition session at the Federation of American Societies for Experimental Biology meeting in Chicago. Working in col­laboration with Dr. W. E. Cornatzer of the University of North Dakota med­ical school, Grand Forks, and Dr. Reu­ben S. Dubois and Dr. K. Michael Hambidge of the University of Colo­rado medical center, Denver, Dr. Evans also finds that metallothionein is involved in copper absorption in the intestine as well as copper stor­age in the liver.

Defect. In Wilson's disease, metallothionein is apparently altered, and the protein has a higher than normal affinity for binding copper, the scientists believe. Only four or five people in a million have Wilson's dis­ease, an inherited defect in copper metabolism that causes toxic copper levels to deposit in the liver and brain. Signs of the disease are a greenish, brownish ring that shows up in the eye, tremors, incoordination, and any manifestation of liver disor­der. The symptoms usually appear in patients during adolescence. Cop­per concentrations in the liver of dis­eased patients range from 100 to 1000 micrograms per gram of wet weight, compared to 5 micrograms per gram of wet weight in normal patients, ac­cording to Dr. Evans. Scientists have tried to explain the genetic abnormal­ity that produces the disease since Dr. Kinnier Wilson first described the syndrome in 1910.

Dr. Bert L. Vallee and his as­sociates at Harvard University med­

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 albumin in the blood to the liver where the metal is bound to metallo­thionein for storage, Dr. Evans says.

Dr. Evans and his associates have isolated metallothionein from the liver of a patient with Wilson's disease and from a control patient. Copper anal­ysis showed that metallothionein from the Wilson's disease patient con­tained a much higher level of copper. Further studies using equilibrium di­alysis indicated that the protein from the patient with Wilson's disease had a higher affinity for copper than did the protein from the control patient.

In the liver of a normal patient, ex­cess copper is excreted into the bile and ultimately eliminated in the feces. In addition, copper is released from the liver to the blood tightly bound to a globular protein, ceruloplasmin, which functions as a circulating reser­voir for copper.

Affinity. However, these mecha­nisms are impaired in the patient with Wilson's disease. As a result of a genetic error, metallothionein in the Wilson's disease patient has a higher affinity for copper. Furthermore, Dr. Evans and his associates think that ceruloplasmin has decreased ability to bind copper.

As the disease progresses, copper accumulates in the liver, and it even­tually exceeds the storage capacity of that organ. The excess copper is spilled over into the blood. Since it is not incorporated into cerulo­plasmin, the copper is deposited in other organs such as the brain.

These experiments may help resolve the controversy regarding the funda­mental cause of Wilson's disease, Dr. Evans says. The work may aid clini­cians in prescribing more effective treatment for the disorder.

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APRIL 26, 1971 C&EN 29