Thank you professor folks Ladies and gentlemen the title of

the discussion as it was given in the program is carcinogenesis chemical physical and biological the

concern for this subject is one of very broad character Yesterday you heard a call for the scientific community to Be concerned with

matters of public interest Well, this one is such a one In our country. Well, first of all, we are all surrounded by What seemed to be a variety of agents in our environment? which

May in fact in some cases we know do have serious consequences for human health these include many of the natural environmental agents such as

Ultraviolet radiation from the Sun or ionizing radiation either from outer space or from the Earth's surface or created by man and of course a wide variety of chemicals some created by the needs of mankind

Some existing in nature in ordinary foodstuffs Now as Professor Fuchs Said a moment ago. It's seems a far cry from the work which he described on

Photosynthesis to the subject upon which I chose to talk today In a sense it is in another sense. It isn't after all the kinds of questions which We sought answers to When we undertook to determine or to try and find out

how a green plant Uses sunshine to manufacture food and material Involved the use of all sorts of all branches of all disciplines of science organic chemistry

physical chemistry physics Biochemistry and biology and later as I have found in the last few years even such matters as Human behavior and economics This subject is a similar one in that sense in that it involves all

kinds of human knowledge and The Specific way in which I came to be involved in this is something that perhaps at least some of you will appreciate

About oh four years ago There came into my laboratory in Berkeley a young man in Italian who Had had a personal what he's an organic chemist and he had a personal experience with the disease of cancer as a result of that he

applied for and received a postdoctoral fellowship from one of the private agencies that Exist all over the world particularly dedicated to the problems of cancer

He also was much interested in Working in our Berkeley laboratory Because some of his friends had been there before him and had told him a little bit about the place So when he arrived his fellowship specified that he had to work on cancer

But we had nothing going in the laboratory on this subject Specifically and so we had a conference Erkale and I sat down He came into my office. We discussed what could be done that would fulfill

the requirement of his fellowship and my interests and after some discussion we agreed that the photochemistry of some carcinogenic agents in the environment would do this and That is how in detail

Specific detail we became involved in the problem That was the beginning of it and you see that's the nature of our lives moving from thing to thing from adjustment to adjustment and Expanding always expanding our interests by virtue of the flux of young people

Which goes through our laboratory? It is the thing which if I Hopefully believe is so that keeps us alive in that particular group of workers well now without

Following that line without trying to give you the details of what Erkale did and how it Expanded into what I'm going to discuss today Point out what he did as we go along Now why is this subject relevant as I said earlier there are

carcinogens various kinds of them physical chemical and biological Surrounding us all the time the chemicals which we add to our food Which we use as drugs and with which we are in contact every day Either in our clothing or in our furniture or in our houses

Contained materials some of which might be and in fact are known to induce the appearance of tumors in men the only way in which this kind of relationship is established is by an epidemiological study we don't do experiments with men usually and

It's interesting to remind you that the earliest known such connection Was discovered by a British physician almost 200 years ago in the case of the Chimney sweeps who cleaned out the chimneys of British homes

you know each year they collect a lot of soot from the coal that they were burning and These chimney sweeps would have to go through those chimneys and sweep them out turned out that There was a particular kind of cancer cancer of the scrotum which was very prevalent amongst these people and

Eventually that cancer was traced to a chemical contained in the soot which collected in the chimneys and This is the one in fact or one of them with which I will spend most of my time

There are many other such materials now that we know about that occur in our food or in our environments the the other end of the story in other words, not only do we have a wide variety of carcinogens that is chemicals radiation and

biological materials of most recent of which has And you've heard about it here before are the viruses But the consequences of their action the way in which the carcinogenesis is expressed varies a great deal

Depending upon that part of the body in which the cancer appears. It looks very different from one to another So we have a wide variety of agents Very different in character and we have what looks like a wide variety of Consequences also very different in overt appearance

However, it is my belief and it is going to be the thrust of everything I say this morning that this wide variety of agents act through a single kind of mechanism not one mechanism but a single kind of mechanism and That the wide variety of expressions of the consequences of the biological consequences

Are all of the same general sort involving a change in the genetic content of Cells and the expression of that change of genetic content may be in a variety of forms

But have one thing in common and it is this commonality Between the Inducing agents on the one hand and the consequences on the other That I would like to bring into one story if I can now as an example

well yesterday at lunch I was reminded of a Story or a bit of advice which I frequently find myself induced to give to some students and What I am going to do today

I'm going to say today is really the embodiment of that advice Very often a student will arrive or work in the laboratory and spend his entire time gathering data without Really trying to integrate it saying that well

He doesn't have all of the information and he's going to wait until he has collected it all Before he begins to think about it now. This isn't really the way Science grows it is true that without data science can't exist or can't continue I can't develop but it isn't really the way in which a new

generation of thought is created at least not in my experience and I've I have to tell them a little story and I do it in this way I say to them it is not in order to be creative in science one must

Occasionally not always in fact very seldom get the right answer But occasionally you must get the right answer about how nature is working when you only when you have only half of the data in your hand and half of what you have is wrong and You don't know which half is wrong and then you get the right answer you are doing something creative

And that's what I'm going to do today. I Haven't got all the data some of what I have is wrong, but I don't know which parts of it are wrong but I'm going to try and put it together and

eventually There will emerge from this construction certain kinds of questions which one can put to the experimental questions which one can put to the test and As we put them to the test We will modify our concepts to fit so that the new concept changed by the new information

grows eventually into a correct one well now let us go on with the nature of Chemical carcinogenesis as we understand it today, of course, it will it might change but today there is a basic

unity at least there appears to be a basic unity in the nature in the chemical nature of the substances which are known to be carcinogenic produced cancer either in man or in experimental animals or more recently in

even simpler systems such as cultures of living cells in a bottle in a in a flask from various sorts of animals and still simpler systems in which we test the chemicals on certain relatively purified Enzyme systems which are molecules themselves. And so we have a range of test systems

From the whole animal on the one hand what mankind on the one hand? To animals to tissue cultures to molecules and we do all of them when we look at the collection of

Molecules which we know to be carcinogenic in one way or in one system or another We see that there is some commonality in their construction and the first slide is simply a catalogue of such chemicals All of them have one thing in common. They all produce

Electrophilic bits this carbonium ion this carbonium ion when the carbon to oxygen bond is broken the negative charge remains on the oxygen the positive on the carbon and this carbonium ion then is an Electrophilic agent seeking a high density of electrons and this is true of the all of these first

Four that I've put on the black on the slide machine here. All of them are Alkylating agents electrophilic reagents giving a positive carbon atom even without any interest enzyme interference and the kinds of electron-dense

molecules with which they react are listed over here in this column and for the Those molecules which are which are common in cells the electrophile I should say the nucleophiles are mostly in the nucleic acid bases either in DNA or an ion RNA or In proteins there may be others as well

These are the principal ones that are seem to be involved in the change in the genetic composition of the cell There are a number of other molecules listed in the lower half which by themselves are not Electrophiles at least not from their first look however After they are changed enzymatically

This one by oxidation of this NH bond to an NO H bond which then can be esterified with acetate or sulfate and This now drops off as a negative ion leaving behind a positive nitrogen ion which in turn Can be and is a powerful electrophile just as these carbonium ions are the same sort of system

Carcinogen which used to be used as a food coloring many many years ago It's no longer so used it was found to be a carcinogen of the liver But and it and it's the mechanism of its transformation from this yellow dye

Into a carcinogen is probably very much the same as this one here by an oxidation of the nitrogen To an NO H And then eventually getting into a nitronium ion so that it can attack These cellular nucleophiles the same thing can be said of these nitrogen compounds now down here

There is a very simple symbol PAH meaning polycyclic aromatic hydrocarbons and these require enzyme activation and when they are so activated they also appear to be

Nucleophiles and it is this group of compounds About which I will speak mostly in terms of the chemical consequences That group of compounds the particular example of which is shown in the next slide All are produced are I should say are produced always when any

Combustion of any organic material occurs for example when you burn tobacco in a cigarette Some micrograms of benzo-a-pyrene the one on the left is produced when you burn gasoline in the internal combustion engine some

milligrams of Benzo-a-pyrene are produced and come out the back end of the exhaust pipe When you burn coal in a power plant or in a heating system in a home a good bit of this Polycyclic aromatic hydrocarbon is part of the combustion products and so that particular group of compounds

Finds its way Into our environment in many different ways even when you burn wood Or I should say Agricultural refuse as a result of the harvesting are the pruning of trees

This also is one of the products that comes out in the smoke, and so it is a common environmental component It is perhaps most effective when it is inhaled into the lungs as a result of smoking a cigarette, but Because then it gets right to the sensitive tissues and so the very few micrograms from each cigarette are

Deposited right in the proper place if you like well now the basic Notion about how this material which is a relatively inert it seems to be a relatively inert chemical these

polycyclic aromatic hydrocarbons, how can they become how can they be made to fit into the notion of electrophilic activity which we mentioned a moment ago in the last decade a great deal of work has been done Implicating an enzyme system which is present in all living cells to some extent

But is induced to a higher level in cells and most cells particularly in liver When they are exposed to such materials as this not only this one But other materials of the same kind will do the similar thing

These are called for obvious reasons the Aerial hydrocarbon hydroxylases a group of mixed function oxidases that is enzymes Which will use molecular oxygen to add one of the oxygen atoms?

to the aromatic hydrocarbon and Use two hydrogen atoms from a reduced coenzyme To take care of the other oxygen atom. This is what is meant by mixed function oxidase They are present in most cells and tissues But they are raised to a higher level when such cells and tissues are exposed

to these aromatic hydrocarbons Well now the next slide shows a way in which that happens aromatic hydrocarbon hydroxylase and molecular oxygen and I didn't put on here the reduced pyridine nucleotide which takes the other oxygen atom and

It was proposed that one of these oxygen atoms is Used to hook on to the aromatic hydrocarbon in the number three and four position making this epoxide now That's a very unstable Compound which with a little bit of acid

Opens to give a carbonium ion at one of these positions either that one or that one which then will react To give a hydroxyl and the remaining carbonium ion will then react with the nucleophile Giving the carbon to carbon bond which I mentioned at the beginning

Thus this is proposed as an obligatory intermediate to make this compound into an electrophile which can then be carcinogenic if this reaction occurs a side reaction which destroys this is simple hydrolysis of this epoxide linkage to give a diode

There are many lines of evidence Which indicate that this hydroxylase is indeed? Involved in making this compound into a carcinogen if one blocks this enzyme in some way Either by a chemical agent which blocks it or by a mutation which prevents it from being made

This material then does not become carcinogenic in such cells so the Participation of the hydroxylase seems to be well established however The isolation of such a product has never been achieved and so we don't really know whether this is indeed the way in which the hydroxylase produces its effect and

It was at this point that the student whose story I told you earlier on entered the picture He undertook to examine the reactions of these aromatic hydrocarbons with components of

Nucleic acids and the one he chose was n-methyl cytosine And the next slide shows what he found Instead of finding the hydroxylated compound on the three or four position He found that the reaction took place on the number six position I should say on the four or five position the reaction took place not here

But on the number six position to give this product now This is a photo chemical induced coupling However, the same reaction can be induced with the arrow hydrocarbon hydroxylase and oxygen exactly the same reaction in which one gets this kind of a

Coupling rather than nose on the sixth position or on the one and the three One three or six and not on the four and five and that was his contribution then to this business Well now we have thus Examined the chemical behavior of a number of carcinogens this one in particular

But as yet we have no clue as to what the biological consequences of such an event might be Why if this happens even if it happens? Why should that produce the biological consequences? That we know it produces namely cancer Now here I have to leave chemical carcinogenesis well one I'll say one more word about it

but we're going to leave cars chemical carcinogenesis for a moment and examine what we know about biological carcinogenesis and then after I describe to you what I think is the nature of

biological carcinogenesis We will see if we can't put the two things together and make a single unitary concept of the whole thing Before we leave this chemical reaction let us see if we can imagine What the molecular consequences at least of such a change would be?

Now I'm going to I don't have pictures of it, and I don't have a model It's much too big to carry from Berkeley to Lindau, so I'm going to ask you to imagine This thing most of you have heard of the structure of what the structure of the genetic

material of living things is the nucleic acids either the DNA or the RNA most all mammalian while all animals have DNA as their principal storage material for the genetic information and that this DNA is primarily first constructed as a sequence of

Bases of which that n-methyl cytosine is one or that cytosine is one instead of the methyl group is a ribose sugar there And it is a long string of such bases of which there are four different kinds The cytosine is only one of them and there are three others But all have similar kinds of electro of electron dense materials in them

They wind up in the double helical structure Which you've all heard about in which the bases are stacked like like playing cards one on top of the other Three and a half angstroms roughly three and a half angstroms apart now if this aromatic hydrocarbon were to

slip in between some of these bases and form that kind of a covalent Carbon to carbon bond as it is described here This is a carbon atom and that's a carbon atom if this were to happen in the double helix the Normal helical structure would be distorted

in fact one could not maintain it if that the reaction occurs and The normal helical structure would be distorted so that at least one of the linear Molecules would be looped out It could not fit in the double helix It would have to be looped out as a sort of a loop outside of the double helix at least that perhaps more

That is a molecular consequence of such a reaction and At the end of this conversation I will try to deduce or introduce that again and go from that to the biological consequences of such an event

But before I can do that, we have to examine what we know or what we think we know about the way in which a virus can Induce cancer can change the character of cells to make them into malignant cells Now most of the work we have done in Berkeley at least in my laboratory that is

Has been not with whole animals, although we've done some of that and I will show you the very last slide some of that But mostly it has been done either with enzymes isolated enzymes and more recently with cells either Animals growing in the test tube in a tissue culture and we've examined the behavior of those cells

Under various influences under viral influence under chemical influence and under both In order for you to get some idea of how this is done I want to show you a picture of a tissue culture of mouse cells

which have been infected with a virus and Wherever the virus particles have have been introduced into the cells and have transformed them by whatever Mechanism and we'll come to that in a moment those cells which are transformed show as little piles of cells and the next slide shows that tissue culture in which a

Few of the cells have been transformed on the left We have only a very weak magnification only a tenfold magnification and the dividing line is right here this background is a background of normal cells cells which have not been infected with virus and

Which grow to confluence and then stop growing? That is when they touch each other when all the cells are touching each other they signal each other They send signals to each other in such a way that the growth of the cell stops

however those cells which have been infected and transformed by the virus the sarcoma virus no longer are subject to that control mechanism and They even though they come to confluence to touching each other they keep on growing and pile up On top of each other and these little piles are called foci and there are visible in this picture

one large focus and Another smaller one and still two more smaller ones There are as I think four foci here one large one and three small ones in which the piles of cells are visible

Even to the naked eye one hardly needs a microscope This is the same picture blown up 40 times instead of 10 times this focus plus that one you can see There it is and each one of these little bright spots is a rounded up cancer cell Piling up on top of some more and here you see the the main the big focus which is that one

And then at the edge of the smaller one here This kind of system is one which has been widely used in the last decade to explore chemical viral and radiation Carcinogenesis rather than using whole animals one of the reasons for this is one can grow a million cells on a dish

Only three centimeters in diameter if one had to grow do similar statistics with a million animals Even mice you can imagine what kind of a housing problem that would be well this has Facilitated the work enormously and it is this kind