Report to the Occupational Disease Panel (Industrial Disease Standards Panel) on Occupational Exposure to Benzene and Leukaemia

Jennifer Penney
September 1995

Benzene Occurrence, Production and Use

Benzene is a clear, colourless, highly volatile liquid, which has been commercially produced and used since the 1860's. (Illinois EPA, 1987) It is widely used, ranking among the top ten chemicals produced worldwide. Canada is a major producer of benzene after the U.S., Europe and Japan. (IARC, 1982)

Benzene is produced primarily by the petrochemical and petroleum refining industries. It is a naturally occurring constituent of crude oil and natural gas, ranging between 0.1 and 3.0 percent by volume, depending on the geographical source. Benzene is also recovered as a byproduct of the coking process in steel mills.

Benzene is an important feedstock chemical. Large quantities are used to manufacture other organic compounds, including ethylbenzene, styrene, cumene and cyclohexanol. (OSHA, 1987) Unreacted benzene may be present in chemicals which use benzene as a feedstock.

Historically, benzene has been an important industrial solvent, especially for rubber, inks, lacquers, paint removers, plastics and artificial leather, (Amdur et al, 1991; ACGIH, 1991) although these uses have been phased out in Canada and most of the developed world due to the toxicity of the chemical. Benzene continues to be employed extensively as a laboratory solvent.

Because of its anti-knock properties, benzene-containing substances are added to gasoline as a replacement for alkyl lead compounds. (Amdur et al, 1991) Gasoline contains from less than one to five percent of benzene by volume. (IARC, 1982)

Benzene Exposures

IARC identifies the following major contributors to benzene emissions into air: (1) gasoline production, storage, transport, vending and combustion; (2) production of other chemicals from benzene; (3) indirect production of benzene (e.g. in coke ovens). Coke ovens are a major source of benzene emissions into water as well. (IARC, 1982)

a) Occupational Exposures

Benzene exposures to workers occur in a number of occupational settings. In petrochemical plants, petroleum refineries and coke production, routine exposures occur mainly to unit operators, tankcar loaders and unloaders, laboratory technicians and maintenance personnel. (OSHA, 1987) In tire manufacturing, process operators, workers who store, mix, load and unload solvents, tire builders and tubers, laboratory technicians and maintenance personnel are all exposed. (OSHA, 1987; Runion, 1985) In steel plants, workers in byproduct and benzol plants and in coke ovens have benzene exposures. Relatively high levels of exposure are experienced by workers in bulk terminals who load benzene or gasoline, particularly in top filling operations and barge loading. (Irving and Grumbles, 1979; Runion, 1985; Holmberg, 1985) Mechanics and gas pump attendants are exposed in automotive service stations. (Holmberg, 1985; Akland, 1993)

A relatively new source of occupational exposure results from the removal of underground gasoline storage tanks. (ACGIH, 1991) Regular work with chain saws or other gasoline-powered equipment may also result in significant benzene exposures.

Because of the high volatility of benzene, inhalation is the most important route of exposure. Almost 50 percent of inhaled benzene is absorbed. (Landrigan and Nicholson, 1992) NIOSH estimates that approximately 1% of benzene is absorbed from skin contact. However dermal absorption is enhanced and may approach 5% when skin is cracked, blistered or abraded as in rubber workers engaged in tire building. (OSHA, 1987)

b) Environmental Exposures

Benzene is released to the general environment from auto exhaust, partly from combustion of other aromatic compounds. It is also given off as a vapour from hot engines and fuel tanks. Exposures increase with rush hour traffic, and with the length of time driving. (Wallace, 1989; Holmberg, 1986; Akland, 1993) Benzene concentrations increase not only inside vehicles, but on heavily travelled streets and in parking garages. As self-service gasoline pumping expands, the general population is increasingly exposed to benzene during automobile refuelling.¹

Cigarette smoke, both mainstream and sidestream, is estimated to account for about 50% of non-occupational exposure. Smokers have mean breath concentrations almost 10 times the levels of non-smokers. (Wallace, 1989) Smoking has been weakly associated with an increased incidence of leukaemia in several studies. (Hoffman, 1989)

Emissions of benzene from smoking and from consumer products such at latex paints, marking pens, rubber products and other common materials can build up in indoor air and contribute to low level exposures. Homes adjacent to service stations may have increased exposures. (Akland, 1993)

Benzene is sometimes found in drinking water, primarily as a result of gasoline spills, or seepage from underground gasoline tanks. (Akland, 1993)

The International Regulation of Benzene

Massachusetts was one of the first jurisdictions to set an occupational exposure limit for benzene, establishing a time-weighted average of 25 ppm in 1946. (Infante, 1987) The American Conference of Governmental Industrial Hygienists (ACGIH) recommended a TLV of 100 ppm in the same year. (ACGIH, 1991)

In 1971, the International Labour Organization adopted a Benzene Convention which contained a number of provisions including: prohibition of the use of benzene in certain work processes; substitution with less toxic substances wherever possible; undertaking occupational hygiene measures to assure effective protection of workers; and provision of medical surveillance of exposed workers. (ILO, 1982)

Finland recognized benzene's leukaemic properties in 1973. (Holmberg, 1986) In 1975, the ACGIH proposed classification of benzene as a suspected human carcinogen and reduced its recommended TLV to 10 ppm. In 1976, the National Institute for Occupational Safety and Health (NIOSH) concluded that benzene is leukaemogenic and recommended an occupational exposure limit of 1 ppm. (Infante, 1987) Subsequently, the Occupational Safety and Health Administration (OSHA) proposed a 1 ppm permissible exposure limit, although due to court challenges, this did not come into effect in the U.S. until 1987. In the late 1970's and early 1980's a succession of countries including Australia, Denmark, Sweden, Norway, the Netherlands and Germany, classified benzene as a carcinogen or suspected carcinogen and prescribed restrictive occupational exposure limits and protective measures. (Holmberg, 1986) The International Agency for Research on Cancer (IARC) concluded in 1982 that there is sufficient evidence that benzene is carcinogenic to man.

Ontario passed a Designated Substance Regulation for benzene in 1984, and set the exposure limit at 5 ppm. Although the background research for the regulation discussed benzene's leukaemogenic properties at some length, the regulation did not identify benzene as a carcinogen.

In 1990, the ACGIH changed benzene's classification to confirmed human carcinogen and lowered its recommended TLV to 0.1 ppm.

The Effects of Benzene on Bone Marrow²

Benzene has been internationally recognized as a potent toxin for some time, particularly for its effects on the blood-forming system of the bone marrow. Benzene exposure has been associated with a large number of haematological disorders. (Goldstein, 1977) The classic haematological disorder caused by benzene is pancytopenia, a progressive decrease in all of the circulating formed elements of the blood: erythrocytes, thrombocytes or platelets, and each of the various types of leukocytes. (Goldstein, 1987; Amdur et al, 1991) Benzene can deplete just one or two of these cell lines as well, with a range of effects from the subclinical to fatal conditions. Benzene was first reported to cause aplastic anaemia in 1897. (IARC, 1982) In the following decades, several other acute and chronic effects of benzene exposure were described. (Goldstein, 1977; Snyder R, 1994; Paustenbach, 1993)

Benzene appears to produce its haematopoietic affects by a number of pathways. Several investigators have reported reductions in the number of multipotent haematopoietic stem cells following exposure of mice to benzene. Benzene depresses the in vitro colony-forming ability of the granulocyte/macrophage progenitor cells. The benzene metabolite trans-trans-mucondialdehyde is cytotoxic to human erythrocyte progenitor cells. Benzene also appears to have a cytotoxic effect on marrow cells of intermediate differentiation regardless of cell line, including promyelocytes, myelocytes, and basophilic erythroblasts. (Kalf, 1987)

Benzene also appears to affect the stromal microenvironment of the bone marrow, resulting in a decreased capacity to support the differentiation of stem cells in exposed mice. Benzene metabolites hydroquinone, p-benzoquinone and phenol also decrease the ability of stromal cells to support formation of granulocytic/macrophage progenitors. (Kalf, 1987)

Hydroquinone and p-benzoquinone inhibit proliferation and differentiation of lymphocytes in culture. Hydroquinone and another benzene metabolite, catechol, appear to reduce the number of progenitor B lymphocytes in vivo, and hydroquinone inhibits the marrow formation of lymphocytes in vitro. Exposure to p-benzoquinone also inhibits the proliferation of T cells.

The Association of Benzene with Leukaemia

The first report which linked benzene to a case of leukaemia was published in 1928 by Delore and Borgomano. (IARC, 1982) Since that time, extensive case reporting, epidemiological research, metabolic and pharmacokinetic investigations, chromosome studies and experimental animal studies have elucidated the relationship between benzene and leukaemia. There is now no question that benzene exposures can cause leukaemia. The international scientific community is agreed that benzene causes acute myelogenous leukaemia (AML), also called acute non-lymphocytic leukaemia (ANLL). (IARC, 1982; Holmberg, 1986; Swaen, 1989; Brett, 1989; Austin, 1988; Goldstein, 1989; Kipen and Wartenberg, 1994; Snyder R, 1994; etc.) Two main issues remain to be resolved, however:

a) Case Reports

The case evidence for an association between benzene and leukaemia is substantial. Just two reviews -- Goldstein (1977) and IARC (1982) -- have identified more than 60 published case reports linking benzene and leukaemia and other lymphohaematopoietic neoplasms.⁴ Several of these case reports spurred early studies of benzene-exposed workers, particularly in Italy, Turkey and France. A number of other case reports of benzene-associated leukaemias and associated disorders are mentioned in studies and reviews undertaken since the IARC assessment, although after the late 1970's individual case reports decreased, due to general acceptance of the leukaemogenic properties of benzene.

More than 150 neoplasms are described in the case reports reviewed by IARC and Goldstein. Almost 40% of these cases involved benzene-linked blood disorders which preceded the leukaemia or other lymphopoietic neoplasm. About one-third of the leukaemias are not identified according to cell type. Of the remainder, slightly less than a third are identified as acute myelogenous leukaemia or one of its variants, slightly more than one-tenth are chronic lymphocytic leukaemias and slightly less than one-tenth are chronic myelogenous leukaemias. The remaining benzene-linked cases involve acute lymphocytic leukaemia, myelofibrosis and myeloid metaplasia, Hodgkin's disease, myelodysplastic syndrome, lymphosarcoma and multiple myeloma.

While case reports do not, by themselves, constitute sufficient evidence for determining causality, they play an important role both in providing preliminary associations for further study and analysis, and as supporting evidence where population studies, experimental evidence, metabolic studies and cytogenetic investigations provide for a causal connection between an environmental exposure and disease. The large number of benzene-leukaemia case reports has served both these functions. The benzene association in many of these cases is strengthened by the diagnosis of benzene-related blood disorders, especially pancytopenia or aplastic anaemia, prior to onset of leukaemia.

b) Epidemiology

Many epidemiological studies have been carried out on benzene-exposed workers in shoemaking, rotogravure (printing), petroleum, petrochemical and rubber industries. A number of these studies followed up on case reports. The relationship between benzene and leukaemia has also been investigated in several hospital-based case-control studies. As in much epidemiology, many of these studies suffer from lack of good exposure data, losses of former workers to follow-up, incomplete or possibly faulty diagnoses on death certificates, potentially confounding exposures, and other problems. Several studies used general population incidence rates for comparison, and neglected to take into account the healthy worker effect in analysis of results. Despite these shortcomings, an impressive amount of evidence has been amassed to support the benzene-leukaemia connection.

Benzene exposures of petroleum workers in these studies were generally lower than levels in the Pliofilm plants or among Turkish shoemakers. The authors also note that many workers unexposed to benzene were included in the cohorts studied. The authors conclude that benzene can increase the risk of AML given a high enough exposure over an extended time, but that petroleum workers were not at risk because benzene exposure levels have been reduced below the threshold for leukaemogenesis, which they suggest occurs at 200 to 500 ppm-years of cumulative exposure.

IARC also cited a Swedish case-control study of acute nonlymphocytic leukaemia (Brandt et al, 1978), noting excess histories of exposure to petroleum products among cases. OSHA cited several additional studies of refinery and petrochemical workers, where risks for leukaemia were elevated, including Theriault and Goulet (1979), Hanis et al (1982), Thomas et al (1982), Devine and Barron (1983), Shell Oil (1983), and Wen et al (1983). Devine and Barron reported significantly elevated deaths from leukaemia among utility workers and pipefitters, who experience relatively high intermittent exposures to benzene. Wen et al also found significantly elevated numbers of deaths from leukaemia in a Texas refinery.

c) Chromosome Studies

Benzene has long been known to produce chromosome abnormalities, which point to the mutagenic potential of the substance. (Snyder and Kalf, 1994) IARC reviewed several chromosome studies which were reported up to 1980. These fell into two general categories: (1) patients with a current or past history of benzene induced blood haemopathies; or (2) workers with current or past exposure to benzene but no obvious clinical effects.

The finding of significant increases in chromosomal aberrations in blood and bone marrow and in lymphocytes from benzene-exposed, symptomatic workers, has been confirmed by several other investigations. (IARC, 1982; OSHA, 1987; Snyder and Kalf, 1994) Studies of asymptomatic benzene-exposed workers have also revealed chromosome abnormalities. Among the studies cited by IARC are:

A number of studies of chromosome damage and genotoxicity have also been carried out in animals. Snyder and Kalf cite several studies which show chromosome aberrations (gaps and breaks) in rabbits, mice and rats exposed to benzene. Chromosome damage leading to the formation of micronuclei in erythrocytes has also been seen in mice treated with benzene, as well as benzene metabolites. (Hite et al, 1980; Tunek et al, 1982; Choy et al, 1985; Gad-El Karim et al, 1986; Anwar et al, 1989) Sister chromatid exchanges have also been demonstrated in mice exposed to benzene by inhalation. (Tice et al, 1982; Kligerman et al, 1983, cited in OSHA) Human blood incubated with benzene metabolites also produced sister chromatid exchanges. (Morimoto and Wolff, 1980) Benzene and its metabolites also induced sister chromatid exchanges in T lymphocytes. (Erexson et al, 1985)

Tice found that more bone marrow damage occurred in mice under intermittent exposure conditions, compared to animals which were exposed constantly. (OSHA, 1987) Toft et al (1982) confirmed that short peak exposures increased the proliferation rate of the bone marrow. Several studies cited by OSHA demonstrated chromosomal damage in animals exposed to benzene at very low doses, including:

Benzene does not appear to be a mutagen when examined in the standardized short-term tests for mutagenesis. (Yardley-Jones, 1991; ACGIH, 1991; Snyder and Kalf, 1994) In vitro tests which fail to metabolize benzene in a way consistent with human or other mammal metabolic transformations do not demonstrate the mutagenic properties of benzene. However, the benzene metabolites p-benzoquinone and trans-trans-muconaldehyde do induce chromosome damage in vitro. (Cox, 1991)

d) Metabolic Studies

In his recent review of the biological basis of chemical carcinogenesis, Cox described the known and postulated pathways of benzene metabolism and leukaemogenesis. (Cox, 1991)

Initial metabolism of benzene takes place primarily in the liver, forming benzene oxide, some of which is detoxified to phenyl mercapturic acid and excreted in the urine. The remaining benzene oxide may spontaneously rearrange to form phenol, or be converted to catechol and/or hydroquinone by liver enzymes. Phenol, catechol and hydroquinone are the three principle liver metabolites of benzene. Catechol and hydroquinone are known carcinogens.⁶ (Huff et al, 1989; Cox, 1991)

Hydroquinone and catechol are carried by the blood stream from the liver to bone marrow where they accumulate and can be found in high concentrations many hours after benzene exposure has ended. (Sawahata et al, 1985) In the bone marrow, these metabolites are transformed to p-benzoquinone, o-benzoquinone and trans-trans-muconaldehyde (TTM). P-benzoquinone causes a variety of genotoxic, clastogenic, and cytotoxic effects, including inhibition of cell division, of DNA and RNA synthesis, growth of bone marrow stromal cells, induction of DNA breaks, micronuclei and sister chromatid exchanges, and binding to DNA and protein. (Cox, 1991; Eastmond, 1993) TTM is also a potent bone marrow toxin, and is known to produce many of the toxic and genotoxic effects of benzene.

Several cell populations of the blood-forming system are susceptible to the effects of benzene metabolites, including pluripotential stem cells, haematopoietic stem cells, early progenitor cells at intermediate stages of differentiation in all cell lines (myeloid, lymphoid, and erythroid), and cells of the bone marrow stromal microenvironment, especially stromal macrophages. (Kalf, 1987; Snyder and Kalf, 1994) Erythroid cells appear to be particularly sensitive to benzene.

While no researcher has definitively proved the mechanism(s) by which benzene induces leukaemogenesis, Cox summarizes five plausible hypotheses based on known cytotoxic and/or genotoxic effects of the substance. Several investigators suggest that more than one mechanism and more than one metabolite are probably involved in benzene leukaemogenesis. (Cox, 1991; Goldstein, 1989; Snyder and Kalf, 1994)

1. Benzene metabolites such as TTM or p-benzoquinone initiate cancer by reacting with cellular DNA, creating DNA adducts that represent inheritable, carcinogenic damage to the somatic cell line;
2. The compensating proliferation of stem cells created by cytotoxic effects of benzene metabolites on partially differentiated cells increases the likelihood of carcinogenic damage, assuming that stem cells are at greater risk of carcinogenic damage while they are actively proliferating than while they are in their normal, more quiescent state;
3. Cytotoxic damage to the stromal microenvironment, including stromal macrophages, impairs its ability to regulate stem cell proliferation and distribution. Initiated and/or malignant stem cells are allowed to proliferate uncontrollably;
4. Cytotoxic damage to the immune system, including lymphocytes and stromal macrophages, allows tumour cells that would normally be detected and killed to survive and proliferate instead, leading to a variety of carcinogenic endpoints;
5. Benzene metabolites initiate cancer through their effects on stem cell chromosomes, by activating oncogenes or inactivating suppressor genes. (Cox, 1991)

e) Experimental Animal Evidence

In addition to the cytogenetic studies described in section c) above, a number of experimental studies demonstrating benzene-induced cancer in animals have been reported. In these studies, benzene has been shown to induce leukaemia, lymphatic cancer and several other types of neoplasm in mice and rats. The production of cancer in more than one species and in more than one organ system reinforces the conclusion that a substance is carcinogenic.

Benzene Exposures and Various Haematologic Disorders

There is clear scientific agreement that benzene causes acute myelogenous types of leukaemia. Ironically, scientific unanimity on this issue may be functioning to impede resolution of a broader question, whether benzene causes a range of other types of leukaemia and lymphatic diseases with which it has been associated. For example, several researchers, looking to investigate the effects of low-level exposures to benzene in various industries, appear to have focused narrowly on the incidence of AML rather than incidence of all types of leukaemia, reinforcing the AML-benzene link, but making more difficult the assessment of linkage with other leukaemias and lymphatic disorders.

Similarly, at least two key investigators -- Aksoy and Yin -- have used proportionate mortality ratios of different types of leukaemia in benzene-exposed workers and the general population as a means of supporting the causal relationship of AML and leukaemia. (Aksoy, 1989; Yin, 1989) In some populations of benzene-exposed workers there is a disproportionately high incidence of AML compared to other leukaemias, and disproportionately low incidence of other types.) Interestingly, neither of these investigators argues that benzene causes only AML. Aksoy reviewed several studies of benzene-exposed workers in which AML predominates, together with two studies in which chronic types of leukaemia occur more frequently and suggests factors to explain the differences, Including:

Aksoy noted the effects of benzene on lymphatic tissues and lymphocytes to support his contention that benzene is associated with Hodgkin's Disease (malignant lymphoma). Several studies have also identified an association between benzene exposure and multiple myeloma (Decoufle et al, 1983; Rinsky et al, 1981). OSHA drew attention to studies which linked benzene and chronic myeloid, chronic lymphatic, and acute lymphatic leukaemias among chemical workers exposed to benzene, as well as multiple myeloma, reticulum cell sarcoma, Hodgkin's disease and other lymphoid tissue neoplasms.

On the basis of information combined from epidemiology and case studies, Kipen and Wartenberg (1994) suggested the following hierarchy of associations between benzene and various Haematologic disorders.⁷

Known

Pancytopenia; aplastic anemia
Acute myelogenous leukaemia (and variants)

Suspected

Paroxysmal nocturnal haemoglobinuria
Chronic myelogenous leukaemia
Chronic lymphocytic leukaemia
Multiple myeloma

Reported

Acute lymphoblastic leukaemia
Myelofibrosis and myeloid metaplasia
Non-Hodgkin's lymphoma
Hodgkin's disease
Thrombocythemia

Myelodysplastic syndrome should probably be added to the list of "known" haematologic disorders caused by benzene exposures. Cullen (1994) and Snyder and Kalf (1994) both suggest that myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) represent a continuum that may be induced in normal stem cells or early progenitor cells by the toxic metabolites which result from chronic exposure to benzene. MDS probably precedes leukaemia in a substantial proportion of all cases. (Cullen, 1994) Aberrations involving chromosomes 5 and 7 have been found in many patients with radiation therapy-induced myelodysplasia prior to the development of AML.

Conclusions and Recommendations

Benzene and its metabolites are toxins to almost all cells of the bone marrow. There appear to be multiple mechanisms for benzene-related toxicity. Therefore it is not surprising that a multiplicity of diseases are associated with benzene exposure.

Case reports, epidemiology, chromosome studies, metabolic studies and experimental evidence all support the conclusion that benzene exposures cause pancytopenia, aplastic anaemia, myelodysplastic syndrome and all variants of acute myelogenous leukaemia.⁸ These diseases should be listed on Schedule 3 and presumed to be caused by work-related exposures to benzene unless "the contrary is proved".

Considerable evidence also exists to link benzene to chronic myelogenous leukaemia, chronic lymphatic leukaemia, multiple myeloma and myelofibrosis and myeloid metaplasia. A number of studies also report an association with acute lymphoblastic leukaemia, non-Hodgkin's lymphoma, Hodgkin's disease and thrombocythemia. Because time was limited for producing this report, the evidence which supports each of these associations was not separately analyzed. The Panel may want to pursue these associations further, however. There is certainly enough evidence to sustain a "probable connection" for several of these disorders, and to develop guidelines to assist adjudicators in deciding whether to compensate benzene-exposed workers who are afflicted with one of these diseases.

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Appendix A: Definitions

Acentric: without a centre; in genetics, describes a chromosome fragment without a centromere (constricted portion of the chromosome at which the chromatids are joined) so that a chromosome will not survive subsequent cell divisions

Adduct: an addition product

Aplasia: absence or defective development of cells or tissue

Aplastic anaemia: disease characterized by depression of all circulating blood cells together with necrosis and fatty replacement of the bone marrow

Basophils: phagocytic leukocytes characterized by numerous granules containing heparin and histamine, found mainly in the blood and at the sites of inflammation; involved in some cell-mediated hypersensitivity reactions

Blood dyscrasia: a diseased state of the blood, usually refers to abnormal cellular elements of a permanent character

Clastogenic: chromosome-breaking

Cytotoxic: destructive to cells

Dicentric: in genetics, describes a chromosome with two centromeres (restricted part of the chromosome at which the chromatids are joined)

Dysplasia: abnormality of development; alteration in size, shape and organization of adult cells

Eosinophils: leukocytes characterized by large red granules that contain basic proteins and many degradative enzymes; the function of eosinophils is not well understood, though they appear to play a role in diminishing hypersensitivity reactions and in resistance to some parasitic infections; human tissue eosinophils are found in the lining of the colon and in other parts of the gut

Epigenetic: relating to the regulation of the expression of gene activity without alteration of the genetic structure

Erythrocytes: mature red blood cells whose major function is to deliver oxygen to the peripheral tissues

Fibroblasts: connective tissue cells which form the the fibrous tissues of the body

Genotoxic: damaging to the DNA and therefore capable of causing mutation or cancer

Granulocytes: mature leukocyte which contains abundant granules in the cytoplasm; includes neutrophils, eosinophils and basophils

Haemopathy: any abnormal condition or disease of the blood or blood forming system

Haematopoietic stem cells: also known as multipotent stem cells, which can differentiate to produce lymphocytic, erythroid, granulocyte-macrophage, and megakaryocyte progenitor cells

Haematopoiesis: the process of formation and development of the blood cells

Histiocytes: macrophages (Reed-Sternberg cells are enlarged, multi-nucleated versions of these, found in Hodgkin's Disease)

Hodgkin's disease: a malignant condition characterized by painless, progressive enlargement of the lymph nodes, spleen, and general lymphoid tissue

Hypoplasia: incomplete development or underdevelopment of a tissue

Leukocytes: white blood cells; classified in two main groups, granular leukocytes (neutrophils, eosinophils, and basophils) and non-granular leukocytes (including lymphocytes and monocytes)

Leukoblast: immature granular leukocyte (also called a leukocytoblast)

Leukaemia: see Appendix C

Lymphocytes: white blood cells formed in lymphatic tissue throughout the body and in bone marrow; in normal adults comprise about 22 to 28% of the total number of leukocytes in circulating blood; two major types: T cells regulate the immune response, are involved in cell-mediated immune reactions and induce B cells to produce antibody; B cells produce immunoglobulin and differentiate into the antibody-producing plasma cells

Lymphomas: malignant neoplasms of lymphoid and reticuloendothelial (red bone marrow) tissues, which present as apparently circumscribed solid tumours composed of cells that appear primitive or resemble lymphocytes, plasma cells, or histiocytes; appear in lymph nodes, spleen, or other normal sites of lymphoreticular cells; may invade peripheral blood and manifest as leukaemia; classified by cell type, degrees of differentiation and nodular pattern

Lymphopoiesis: the formation of lymphocytes

Macrophages: scavenger cells arising from monocytic stem cells, widely distributed in the body and varying in form, structure and motility; attack microorganisms and neoplastic cells and remove foreign material from tissue

Megakaryocytes: also called thromboblasts; a large cell normally present in the bone marrow, not in circulating blood, which gives rise to platelets

Micronucleii: detached chromosomal fragment that remains as micronucleus in mature red cells after extrusion of the nucleus; indicative of chromosome breakage

Mitosis, mitotic: referring to cell division

Monocytes: mononuclear phagocytic leukocyte, formed in the bone marrow, circulate in peripheral blood and then enter tissues such as the lung and liver where they develop into tissue macrophages

Multiple myeloma: also termed plasma cell myeloma or myelomatosis, multiple myeloma is a malignant neoplasm characterized by the poorly controlled growth of a single clone of plasma cells

Myelocytes: young cells of the granulocytic series, occurring normally in bone marrow but not in circulating blood

Myelodysplastic syndrome (MDS): a group of disorders of the bone marrow with the following characteristics: anemia; abnormal numbers [+/-] of platelets and leukocytes with morphologic abnormalities, but not blasts; a hypercellular bone marrow with abnormal erythrocytes and often abnormalities in other cells; abnormalities on marrow cytogenetic studies suggesting the presence of a malignant clone. Sometimes called preleukaemia. Leukaemia develops in 10 - 20% of cases studied.

Myelofibrosis with myeloid metaplasia: a clonal stem cell disorder resulting in marrow fibrosis associated with anaemia and often high white blood cell counts.

Myelogenous: produced by or originating in the bone marrow

Myeloid: pertaining to or derived from the bone marrow

Myeloid fibrosis: also called myelofibrosis; formation of fibrous tissue in the bone marrow as part of reparative or reactive process

Myeloproliferative disorders: neoplastic disorders of the multipotent haematopoietic stem cell, including chronic myelogenous leukaemia, myeloid metaplasia with myelofibrosis, polycythemia vera, and essential thrombocytosis

Neutrophils: mature cells in the granulocytic series, formed in the bone marrow and released into the circulating blood where they normally represent from 54% to 65% of the total number of leukocytes and play an important role in the body's defense by ingesting and destroy microorganisms and foreign particles

Non-Hodgkin's lymphoma: neoplastic proliferation of lymphoid cells that usually disseminate throughout the body; (also called lymphosarcoma and reticulum cell sarcoma)

Oncogenes: transforming genes, found in certain retroviruses, which may transform a cell to a neoplastic phenotype

Pancytopenia: pronounced reduction in the number of erythrocytes, all types of white blood cells, and the blood platelets in the circulating blood

Phagocytic: relating to the engulfing and destruction by cells of foreign particles, microorganisms or other cells

Plasma cells: mature, antibody-producing cells which are derived from B lymphocytes

Platelets: disk-shaped structure found in the blood; known chiefly for role in blood clotting but also involved in the immune response

Pluripotent or pluripotential stem cell: the root cell in the haematopoietic system, capable of reproducing itself to maintain the stem cell compartment of the bone marrow, or differentiate to produce colonies of erythroid, granulocytic, megakaryocytic and lymphoid cells

Promyelocytes: precursor in the granulocytic cell series, between myeloblasts and myelocytes, containing few as yet undifferentiated cytoplasmic granules

Protooncogenes: preexisting gene which appears to have a role in normal cellular physiology and is often involved in normal cell growth or proliferation

Reticuloendothelial system: also called macrophage system; an important bodily defense mechanism, composed of highly phagocytic cells having both endothelial (lining) and reticular (net-like) attributes; these cells include macrophages lining the lymph sinuses and the blood sinuses of the liver, spleen, and bone marrow, reticulum cells of lymphatic tissue, tissue macrophages and circulating monocytes;

Sinusoid: a form of terminal blood channel found in the haemolymph glands and other organs

Sister chromatid exchange (SCE): exchange of segments between the two chromatids or arms of a chromosome; indicates that the DNA or genetic material of a cell has been altered by some genotoxic agent; believed

Somatic cells: cells of the organism other than the germ cells

Stroma, stromal: relating to the framework, usually connective tissue, of an organ, gland or other structure; in the bone marrow, a framework of fat cells, fibroblasts, macrophages, blood vessels and sinusoids that play a nutritive role in haematopoiesis

Thrombocytopenia: decrease in the number of blood platelets

Thrombocythemia: a condition characterized by a fixed increase in the number of circulating blood platelets

Zymbal gland: gland in rodents with no counterpart in human anatomy

Appendix B: Bone Marrow, the Formation of Leukocytes and Lymphocytes, and Benzene

The average adult has approximately 1.7 L of bone marrow, which contains 1012 progenitor cells which replenish the body's red and white blood cells and platelets. The marrow ensures the production of a variety of blood cell lines to meet different physiologic needs, creating a generational hierarchy of cells that have differing capacities for self-renewal, growth, differentiation and maturation. (See Figure 1 on the following page.) These cells grow and mature on a framework of fat cells, fibroblasts, macrophages, blood vessels and specially designed sinusoids. This marrow network provides the factors which control the growth and differentiation of the marrow progenitor cells. Normally, only mature cells can cross the sinusoidal wall and enter circulating blood.

At the top of the hierarchy is the pluripotent stem cell. The adult human may have only 106 to 108 of these stem cells.(Schrier) The pluripotent stem cell may undergo self-renewal or differentiate to produce haematopoietic stem cells or B and T lymphoid precursor cells. (B and T cells are, however, mainly produced in lymph tissue, not bone marrow.)

The haematopoietic daughter cells of the pluripotent stem cell have a limited capacity for self-renewal, and also differentiate to produce pure or mixed colonies of erythrocytic, granulocytic or megakaryocytic cells. The granulocytic progenitor cells may differentiate again to produce cell lines leading to the production of mature basophils, neutrophils, macrophages and eosinophils.

If demands for both renewal and differentiation are too great, the stem cells will undergo self-renewal exclusively, reducing the level of precursor cells and mature erythrocytes, leukocytes and platelets in the body. If the numbers of stem cells are reduced to some critical level, the marrow fails and the syndrome of aplastic anaemia results.

Appendix C: Leukaemia

Leukaemia is a progressive, malignant disease of the blood-forming organs, characterized by the proliferation of abnormal haematopoietic cells in the blood and bone marrow. Cells show a decreased capacity for normal differentiation, expand at the expense of normal cell lines and impair normal myeloid or lymphoid cell growth. This frequently leads to death by infection or haemorrhage. Diagnosis is based on two findings: the presence of abnormal cells in peripheral blood and the presence of abnormal cells replacing normal bone marrow elements.

These neoplasms arise from a the malignant transformation of various cell lines which proliferate initially in the bone marrow and then spill over into the peripheral blood. The particular type of disease which develops depends on the point in the cell line at which transformation occurs and further development or maturation is blocked. (See Figure 1 in Appendix B.) Kipen and Wartenberg describe it this way:

"(D)evelopmental blockade at the pluripotent stem cell level causes aplastic anaemia... If the block occurs a stage later, acute myelogenous leukaemia (AML) results. Later stages of maturation arrest result in progressively more differentiated cells up through acute megakaryocytic leukaemia. In chronic myelogenous leukaemia (CML), relatively mature, although dysfunctional, cells are produced by an incomplete maturation block; however, these cells typically become unstable due to subsequent development of an early block with proliferation of promyelocytes and blasts.

"Acute and chronic lymphatic leukaemias (ALL, CLL) are disorders of committed lymphopoietic stem cells, originating in lymphoid precursors of bone marrow, thymus, and lymph nodes." (Kipen and Wartenberg, 1994)

Leukaemia is classified clinically on the basis of:

There are several classification systems for leukaemia. Most of these categorize leukaemias into four major groupings:

1. Acute myelogenous leukaemia or AML (also known as acute myelocytic leukaemia or acute non-lymphocytic or ANLL), of which there are several sub-types, including:

It is sometimes difficult to distinguish these different cell types from each other, or the undifferentiated type of leukaemia from the adult form of acute lymphoblastic leukaemia. (Champlin, 1987)

AML is the most common acute leukaemia among adults. Abnormal myeloid precursors infiltrate and replace bone marrow tissue.

Many patients develop AML after a preleukaemic syndrome, or myelodysplastic syndrome (MDS) usually characterized by anaemia, thrombocytopenia (decrease in platelets) and sometimes granulocytopenia (decrease in basophils, eosinophils and neutrophils), associated with a dysplastic bone marrow. Although it may not progress to AML, MDS is often fatal. (Cullen, 1994)

Several environmental exposures are suspected to cause AML, including ethylene oxide and ionizing radiation. Styrene, 1,3-butadiene, vinyl chloride, paints and nitrites have also been linked to AML. (Kipen and Wartenberg, 1994)

2. Acute lymphocytic (lymphoblastic) leukaemia (ALL)

Although ALL can also be subdivided by cell type, these sub-classifications are not used in the benzene-related leukaemia literature, and so will not be elaborated on here.

In ALL, abnormal lymphoid precursors infiltrate and replace bone marrow and lymphatic tissue.

ALL is primarily seen in children and to a lesser extent in adolescents, although a late peak in incidence occurs after age 60. Several maternal and paternal occupations have been linked to ALL in children, especially in the nuclear industry. (Kipen and Wartenberg, 1994)

3. Chronic myelocytic (myeloid, myelogenous) leukaemia (CML)

CML is one of several diseases classified as a myeloproliferative disorder, resulting from transformation of the multipotent haematopoietic stem cell. It is characterized by an accumulation of both immature and mature granulocytic cells, suppressing normal maturation of granular leukocytes and erythrocytes. It is often discovered as a result of an elevated white cell count in the absence of symptoms. Ten to 15% of patients also have marrow fibrosis, creating difficulties in differentiating CML from myelofibrosis with myeloid metaplasia. (Kipen and Wartenberg, 1994)

CML has been linked with benzene exposures.(Goldstein, 1989; Vigliani and Forni, 1976; Kipen and Wartenberg, 1994) Rubber workers and electrical workers have been shown to be at increased risk for CML. (Kipen and Wartenberg, 1994)

4. Chronic lymphocytic leukaemia (CLL)

CLL is mainly a disease of later life and is characterized by high circulating levels of mature-looking lymphocytes, usually neoplastic B lymphocytes. (Champlin) It has a more favourable prognosis than the other leukaemias and often requires no therapy.(K & W) CLL is described as a "variant of well-differentiated non-Hodgkin's lymphoma". (Kipen and Wartenberg, 1994) CLL has not been strongly associated with benzene exposures.

Because classification systems and diagnostic techniques have changed substantially over the last several decades, complicating the process of linking different types of leukaemia with specific environmental agents. Kipen and Wartenberg elaborate on this issue:

"A number of caveats are important. Nomenclatures within the LH neoplasms have changed frequently over the last 3 decades. The International Classification of Diseases (ICD) and clinical coding practices are not well synchronized with one another nor with trends in clinical diagnostic practice. Thus, deaths due to myelodysplastic syndrome (MDS) are sometimes classified as a type of AML and sometimes separately, and within NHLs (Non-Hodgkin's Lymphomas) there have been numerous shifts in nomenclature, whereas the ICD has retained the older terminology. Because in any population LH neoplasms are relatively rare, such inconsistency influences the relative occurrence of specific LH cancers, and thus impedes epidemiologic recognition and investigation of etiologic associations. For this reason, the total number of LH neoplasms or leukaemias based on death certificates usually is reliable, because LH tumours rarely are classified outside of the overall leukaemia and lymphoma rubric, but specific cell types cannot be reliably ascertained from death certificates.

"For this reason, in many epidemiologic analyses, the leukaemias are considered as one... The paradox is that, to the extent that the tumours are distinct entities with distinct sets of causes, imprecision in classification makes etiologic associations more difficult to observe with consistency, whereas to the extent that one cause may result in multiple distinct histologic abnormalities, splitting arbitrarily reduces epidemiologic power and the opportunity to observe statistically significant associations."

Appendix D: Case Reports Linking Benzene, Leukaemia and other Lymphopoietic Neoplasms

The descriptions for most of these case reports are derived from reviews by IARC (1982) and Goldstein (1987).

Endnotes

¹ Recently, Federal Environment Minister Sheila Copps announced plans to limit benzene in gasoline to 1% by volume, and to require the natural gas industry to install technology to burn or recapture benzene from gas before it is shipped by pipeline. This should decrease automobile and gasoline pumping exposures. (Westell, 1995)

2 For a description of the normal functioning of the bone marrow, and the cell lines produced in bone marrow, see Appendix B.

3 See Appendix C for a discussion of the different types of leukaemias and other relevant lymphohaematopoietic disorders.

4 Most of these case reports are described in Appendix D.

5 Wong and Raabe argue that leukaemia is a group of distinct malignancies which arise from changes in committed stem cells, rather than in the pluripotential stem cell precursor, and therefore must be investigated independently. Further discussion of this issue occurs later in this paper.

6 Catechol causes glandular stomach cancers. Hydroquinone elevates the incidence of leukaemia in female rats and liver adenomas in female mice.

7 Kipen and Wartenberg actually adapted this table from Goldstein (1989).

8 While aplastic anaemia occurs usually as the result of fairly high exposures, leukaemia has been shown to occur as a result of exposures below the current Ontario TWAEV. (Infante, 1989) Several investigators have found that increased risk is associated with cumulative exposure, whereas others have postulated that intermittent or peak exposures are more important risk factors. With respect to latency, there appears to be a minimum lag period of 3-5 years from first exposure and a peaking of AML incidence within 8-10 years for individuals who develop therapy-related leukaemia. In the Pliofilm cohort, time from initial exposure to death due to a haematological neoplasm ranges from 3.5 to 37 years. (Goldstein, 1989)