Is dothistromin a human health risk? Christmas tree may not be....

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http://www.blackwell-synergy.com/links/doi/10.1111/j.1439-

0329.2004.00356.x/full/

3.3 Is dothistromin a human health risk?

The structural similarity of dothistromin to aflatoxins prompted

serious concerns about the health risk associated with Dothistroma

needle blight, especially for forest workers. Aflatoxin B1 is the

most potent naturally occurring carcinogen known ( Squire 1989) and

is classified as a group I carcinogen by the International Agency

for Research on Cancer. It is also teratogenic (causing deformities

in embryos) and toxic. Cancer research with animals showed that a

minimum dose of 50 ng aflatoxin B1/kg body weight/day is necessary

to produce tumours, whilst toxicology studies with humans exposed to

dietary aflatoxins have suggested the acute lethal dose of aflatoxin

B1 for adults is 1020 mg ( Elliott et al. 1989; and Klich

2003). Aflatoxins are activated to more potent forms by microsomal

enzymes. Cytochrome P450 enzymes convert the unsaturated bisfuran

ring of aflatoxin B1 to the highly carcinogenic 8,9-epoxide form

that can bind to DNA ( Eaton and Gallagher 1994). Recent work by our

group in collaboration with Dr D. Bhatnagar's aflatoxin group (New

Orleans SRRC, USDA) has shown no evidence for aflatoxin production

by D. pini but the acute toxicity and potential genotoxicity of

dothistromin remains a concern.

Table 3 summarizes dothistromin toxicology studies that have been

carried out with a wide range of organisms and cell types. The

reader is also referred to the comprehensive summary of published

and unpublished work on this topic by Elliott et al. (1989).

Toxicity has been measured by growth inhibition, cell lysis and

mitotic index. Dothistromin is toxic to P. radiata tissue at low

concentrations, but is also highly toxic to a range of bacterial,

fungal, plant, animal and human cells (Table 3). In most studies

light was required for toxicity, but the brine shrimp Artemia salina

was susceptible to the effects of dothistromin without light

activation ( Stoessl et al. 1990). Compared with aflatoxin B1,

dothistromin appeared to have higher toxicity to Bacillus

megaterium, with concentrations of 510 g/ml aflatoxin B1 but only

0.51.0 g/ml dothistromin required to completely inhibit growth (

Harvey et al. 1976). However, when a direct comparison of these

toxins was made with human lymphocytes using mitotic index as a

measure of toxicity, there was little difference between them (

Ferguson et al. 1986), although when aflatoxin was metabolically

activated it caused a significant depression of mitotic index

(suggesting higher toxicity) compared with that seen with

dothistromin.

Does dothistromin cause damage to genetic material and is it a

carcinogen? There have been no epidemiological studies reported to

date that suggest it is a carcinogen: reviews of cancer incidence in

forestry workers showed no evidence to connect dothistromin with

lung cancer ( Elliott et al. 1989). However, there is evidence that

dothistromin is a weak mutagen and clastogen. Dothistromin showed

significant mutagenic activity in an Ames assay ( Ferguson 1986;

Elliott et al. 1989) and in a screen for specific induced mutations

in Chinese hamster fibroblasts (McLarin and Ferguson 1985) but

mutagenic activity was weak compared with aflatoxin B1. In both

cases, it appears that metabolic activation enhanced mutagenicity

but unfortunately the details of these studies were not published.

Clastogenic activity of dothistromin has also been demonstrated with

tests for chromosome aberrations and sister-chromatid exchange (SCE)

in human cells and with micronucleus assays in rodent cells (McLarin

and Ferguson 1985; Ferguson et al. 1986; Elliott et al. 1989;

Skinnider et al. 1989). Chromosome aberrations caused by

dothistromin were mostly simple gaps and deletions and were not

affected by metabolic activation, whilst aflatoxin B1 (with

metabolic activation) induced a higher level of aberrations at

equivalent doses, with more complex exchange type aberrations.

Similarly the effect of dothistromin as demonstrated by the

micronucleus assay with Chinese hamster cells, was weak compared

with aflatoxin B1 and was not dependent on metabolic activation

(McLarin and Ferguson 1985). In a study of SCE induced by

dothistromin in human cells, Skinnider et al. (1989) made the

interesting observation that purified lymphocytes show a greatly

enhanced sensitivity to dothistromin compared with lymphocytes in

whole blood (a threefold increase in SCE with only 1/10 the

concentration of dothistromin). This effect was not seen with the

mitomycin control and it was therefore postulated that dothistromin

may be 'neutralized' by some component in whole blood. There is

clearly a lot more to discover about the complexities of how

dothistromin might act in vivo. The only in vivo study on mammals

reported in the literature showed an equivalent clastogenic effect

of dothistromin and aflatoxin ( Elliott et al. 1989) although the

increases in clastogenicity seen with these toxins were not

significantly greater than a toxin-free control. The authors

attributed this lack of statistical significance partly to the

genetic diversity of the mice used in the study.

Because dothistromin is highly toxic to a wide range of cell types

at low doses it appears that there is a narrow difference between

effective doses for mutagenicity/clastogenicity and toxicity, with,

in effect, cells being killed before their genetic material is

damaged. Ferguson et al. (1986) wrote that the 'toxicity may be

masking any major potential for clastogenicity by dothistromin'. In

summary, there is evidence that dothistromin is a weak mutagen and

clastogen in mammals, although at higher doses there is more likely

to be toxicity to cells rather than an increased risk of genetic

damage.

Research from the New Zealand Forest Research Institute (NZFRI) and

Department of Health indicated low levels (<7 ng/ml) of dothistromin

in air and water from the catchment area of forests with Dothistroma

blight (documented by Elliott et al. 1989). Dothistromin levels are

higher in wet periods than in dry, and are higher at certain times

of the year such as during the summer when the fungus is most active

( Briggs 1985; Ferguson 2002). The exposure of forestry workers

involved in thinning, low pruning or final clear felling of pine

trees with 2340% mean Dothistroma disease rate was monitored by

measuring dothistromin levels in the air, on clothing and on skin (

Briggs 1985). Although low but quantifiable levels of toxin were

detected, there was a wide variation even between workers carrying

out the same job on the same day, for example, air filtered from the

breathing zone of two workers engaged in pruning contained <0.0009

and 0.1493 ng/ l dothistromin. This variation could be accounted

for by the collection of a few particles (such as needle fragments)

containing relatively high levels of dothistromin, rather than the

collection of many particles with low toxin content. Despite the

generally accepted low risk to forestry workers nonetheless it is

prudent to limit exposure. The New Zealand Dothistroma Control

Advisory Committee ( Anonymous 1986) advised the following

(reproduced with permission from the NZ Farm Forestry Association):

In dry conditions there is a negligible risk of exposure to

dothistromin. In wet stands the recorded incidence of dothistromin

was higher but still too low to confirm a real exposure risk to

workers.

Wherever practical, work should be scheduled away from infected

stands during wet weather.

It is important to ensure workers wear protective clothing when

working in infected stands in wet weather.

It is important to correctly implement the spray programme

recommended by the Disease Control Advisory Committee (all stands

with disease level above 15% and in which workers will be operating

over the next 12 months are to be sprayed).

Further advice given by Briggs (1985) includes:

Forestry workers should wear clothing that does not collect

dothistromin-containing particles: cotton is preferable to wool.

Work clothes should be washed frequently and exposed areas of the

body such as hands, face and hair should be kept clean to prevent

accumulation of dothistromin-containing particles.

Disposable dust masks should be supplied for use by workers.

However, note that, in New Zealand, after decades of coping with

Dothistroma needle blight in commercial forests these practices are

not adhered to rigorously nowadays as there has been no evidence of

detrimental effects on forestry workers.

http://www.biomedcentral.com/news/20041223/01/

December 23, 2004 Previous | Next

Spreading fungus, not cheerIncrease of red band needle blight means

that Christmas tree may not be quite so pretty | By Branwen

Not everyone wants a real Christmas tree—after all, they only last

one season and make a mess with all that dropped foliage—but many do

like to have the choice. Unfortunately, levels of Dothistroma pini,

a toxic fungus that causes red banded lesions on pine needles and

premature defoliation, could affect such decisions.

A number of countries in the Northern hemisphere, including the

United Kingdom, France, and Canada are reporting an upsurge in the

severity and distribution of D. pini infection. Milder and wetter

weather conditions are partly to blame, as the spread of this

pathogen is favored under moist, warm, light, and sheltered

conditions.

Brown, who heads the Dothistroma Project at Forest Research,

UK, told The Scientist: " This disease has become particularly

noticeable over the past 2 years and has apparently been increasing

in frequency for 5 to 6 years. In the 2002–2003 period, we found

over a quarter of the trees were suffering serious defoliation in

the East Anglia forest district, where large numbers of the highly

susceptible Corsican pine are planted. "

New Zealand and Australia have been coping with widespread D. pini

infection for decades and have been quite successful in protecting

their pine forests. The fungus rarely kills the tree, but the

percentage of pine needles infected is directly proportional to loss

of wood yield, so D. pini can have a severe economic impact.

One new approach that could be fruitful is to genetically engineer

trees that inactivate, remove, or prevent synthesis of toxin by the

fungus. The toxin, dothistromin, is thought to be the primary cause

of red band needle blight and is structurally related to the potent

carcinogen aflatoxin, produced by some fungi from the genus

Aspergillus. This similarity has enabled scientists to identify and

isolate key genes involved in the synthesis pathway.

Rosie Bradshaw, from Massey University in New Zealand, is one of a

small number of researchers worldwide who is focusing her efforts on

taming the feisty fungus. Her team has knocked out some of the genes

that are essential for synthesizing dothistromin. " We now have the

means to determine if dothistromin is responsible for pathogenicity

and are currently trying to infect whole pine needles, before trying

seedlings in the lab. We also have GFP reporter strains, so we'll be

able to track where and when the dothistromin genes are expressed.

If dothistromin production is essential, it will be a good target

for disease control, " she told The Scientist.

One option would be to use non–toxin-producing strains as biocontrol

competitors with the toxigenic fungi in the field. Others include

engineering trees that express dothistromin-specific antibodies in

the needles or targeting destruction of the toxin in the plant

tissue by enzymatic means. Another approach is the deployment of

toxin transporter genes into host plants. These transporters are

normally used by the fungus to allow secretion into plant tissue and

to provide a self-protection mechanism against the effects of its

own toxin. Bradshaw has already isolated a candidate dothistromin

transporter from D. pini that could be used in this way.

Bradshaw acknowledged that genetic modification of trees is not

necessarily the best option for disease management. " But in the long

term, it may well be our detailed knowledge of the molecular biology

of the pathogen and its toxin that will provide the basis for

sustainable control, " she said.

So, where does that leave the Christmas shoppers who want nothing

more than a glass of mulled wine and a pine tree to decorate? " I

would think people probably wouldn't buy a tree with Dothistroma

simply because they aren't very pretty—unless you're a pathologist, "

said Woods, a regional forest pathologist in British Columbia,

one of the areas worst affected. " That said, I did notice an

advertisement for Wal-Mart of people picking out pine Christmas

trees that all looked pretty badly defoliated by a foliar disease,

and they only had the current years growth on them... Seriously! But

I guess only a pathologist would have seen that. "

Links for this article

R.E. Bradshaw, " Dothistroma (red-band) needle blight of pines and

the dothistromin toxin: a review. " Forest Pathology, 34:163-85, June

2004.

http://www.blackwell-synergy.com/links/doi/10.1111/j.1439-0329.

2004.00356.x/full/

Rosie Bradshaw

http://imbs.massey.ac.nz/Staff/bradshaw.html

P. Smaglik, " Biocontrols: an alternative to pesticides? " The

Scientist, October 12, 1998.

http://www.the-scientist.com/1998/10/12/1/3