Porphyric Pesticides - American Chemical Society

Chapter 17

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Effect of Protoporphyrinogen Oxidase Inhibitors on Mammalian Porphyrin Metabolism J. Krijt, M. Vokurka, J. Sanitrák, and V. Janousek Department of Pathological Physiology, First Medical Faculty, Charles University, C Z 128 53 Prague, Czech Republic

The herbicide fomesafen (100 ppm) significantly increased the porphyrin content of mouse liver and bile when fed to male ICR mice for 10 days. Liver porphyrin concentration increased from 0.7 to 1.6 and bile porphyrin concentration from 7.4 to 38.2 nmol/g wet wt. Feeding of 2500 ppm of fomesafen in the diet for 5 months increased the liver porphyrin content from 1.0 to 138.2 nmol/g, with uroporphyrin and heptacarboxylic porphyrin forming the major porphyrin fractions. Fomesafen, oxadiazon, and oxyfluorfen also caused in vitro porphyrin accumulation in experiments with mouse hepatocyte cultures and HepG2 cells. However, it is concluded that these herbicides probably pose no toxicological hazard when properly used.

Many xenobiotics are capable of disrupting the porphyrin biosynthetic pathway in vivo. A classic example is the fungicide hexachlorobenzene, which caused a massive outbreak of cutaneous porphyria in Turkey. Other polyhalogenated aromatic hydrocarbons like 2,3,7,8 tetrachloro-p-dioxin (TCDD), octachlorostyrene, and polychlorinated and polybrominated biphenyls have been documented to induce porphyria in experimental animals and, in some cases, in humans (7,2). A l l these polychlorinated aromatic hydrocarbons act through inhibition of uroporphyrinogen decarboxylase. Another porphyrogenic chemicals, the ferrochelatase-inhibiting drugs griseofulfin and D D C , cause a dramatic increase in hepatic protoporphyrin levels when fed to experimental animals (3). Thus, the porphyrin biosynthetic pathway clearly can become an inadvertent target of industrial chemicals or byproducts. Recently, a new group of potentially porphyrogenic chemicals has been identified. Photobleaching herbicides like diphenyl ethers or oxadiazon have been demonstrated to inhibit the enzyme protoporphyrinogen oxidase (4,5,6). In addition, some other structurally unrelated herbicides are also capable of

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Duke and Rebeiz; Porphyric Pesticides ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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PORPHYRIC PESTICIDES

protoporphyrinogen oxidase inhibition (7). Since these herbicides are in widespread use, their possible adverse effect on the heme biosynthetic pathway in mammals should be carefully considered. Increased Porphyrin Concentration in Mouse Tissues Induced by Short Term Feeding of Photobleaching Herbicides. Addition of 2500 ppm of fomesafen, oxyfluorfen, or oxadiazon to the diet increased the porphyrin content of liver, bile, and feces when fed to male mice of the ICR strain for 10 days (Table I). The pattern of liver, bile, and fecal porphyrin accumulation induced by these herbicides was determined by high performance liquid chromatography of the porphyrin methyl esters. In all cases, protoporphyrin and uroporphyrin constituted the major liver porphyrin fractions, while protoporphyrin and coproporphyrin predominated in the bile and feces. The same pattern of fecal porphyrin excretion is also found in the human disease variegate porphyria, which is caused by an inherited deficiency of protoporphyrinogen oxidase (3). Urine of oxadiazon-treated male Wistar rats (lOOOppm, 6 days) contained elevated amounts of uroporphyrin, coproporphyrin and porphobilinogen. This excretion pattern again parallels the findings in variegate porphyria.

Table I.

The Effect of Protoporphyrinogen Oxidase-inhibiting Herbicides on Porphyrin Accumulation in Male I C R Mice

Treatment

Liver Porphyrin Content (nmol/g wet wt)

Bile Porphyrin Content (nmol/g wet wt)

Control Fomesafen Oxyfluorfen Oxadiazon

1.2 3.6 15.8 29.5

9.2+ 5.1 134.5 + 33.6 340.3 + 123.3 481.2 + 170.1

+ + + +

0.1 0.1 3.4 2.1

Fecal Porphyrin Content (nmol/g dry wt) 28.9 296.7 543.0 917.0

Mice were fed 2500 ppm of the herbicides in the diet for 10 days (n=3). Porphyrins were extracted and esterified by methanol/sulphuric acid (95:5 v/v), and quantified on a spectrofluorimeter (407/607 nm) using protoporphyrin dimethyl ester as standard. Fecal porphyrin content was determined in pooled samples.

The porphyrin accumulation induced by high dose, short term fomesafen treatment is reversible. Pretreatment of male ICR mice with 5000 ppm of fomesafen in the diet for 10 days resulted in increased porphyrin concentrations in liver, bile and feces (Table II). After further 5 days without fomesafen treatment, the fecal and bile porphyrin content returned to normal levels, and liver porphyrin content was only slightly elevated.


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Table Π .

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Mammalian Porphyrin Metabolism

The Effect of a 5-day Recovery Period on Increased Porphyrin Levels Induced by Fomesafen Pretreatment Porphyrin Concentration (nmol/g)

Treatment Liver Control diet, 10 days Fomesafen, 5000 ppm, 10 days Fomesafen 10 days, control diet 5 days

Feces

Bile 4.3

34.5

80.1 ± 1 5 . 3

527.3

6.4+

1.2 + 0.3 6.0 + 2.3

9.6+

1.8 + 0.3

40.1

3.2

Fecal porphyrins were determined in pooled samples, η = 3 .

As indicated in Table I, a high dose of fomesafen produced only modest changes in liver porphyrin concentration. However, subsequent experiments demonstrated that lower doses of fomesafen are sufficient to induce a statistically significant increase in liver porphyrin content (Table III).

Table Π Ι . Liver and Bile Porphyrin Concentrations of Male I C R Mice Fed 100 ppm of Fomesafen in the Diet for 10 Days Treatment

Relative liver weight (%)

Control diet Fomesafen 100 ppm

5.0 + 0.1 7.2 + 0.4

Liver porphyrin content (nmol/g)

7.4 + 4.6

0.7 + 0.1 a

1.6 + 0.2

Bile porphyrin content (nmol/g)

a

38.7 + 8.9

a

"Statistically different from control values, ρ < 0 . 0 5 (η=3).

Elevated Levels of Uroporphyrin and Heptacarboxylic Porphyrin Induced by Long Term, High Dose Treatment with Diphenyl Ether Herbicides. When diets containing high concentrations (2500 or 5000 ppm) of fomesafen were fed to ICR mice for a sufficiently long period, the pattern of liver porphyrin accumulation gradually changed. In some animals, highly carboxylated porphyrins, i.e. uroporphyrin and heptacarboxylic porphyrin, formed the major liver porphyrin fraction after 3 to 5 months of treatment (Table IV). The total liver porphyrin content of these animals was dramatically increased but showed marked individual variation. Interestingly, the same pattern of porphyrin


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accumulation, as well as the wide scatter of individual values, is typically observed after long term treatment with polyhalogenated aromatic hydrocarbons like hexachlorobenzene, polychlorinated biphenyls, or T C D D (1,3). This unexpected accumulation of highly carboxylated porphyrins after long term treatment with a protoporphyrinogen oxidase inhibitor was further confirmed in one experiment with the commercial herbicide formulation Blazer, containing acifluorfen-sodium. Addition of Blazer (corresponding to 2500 ppm of acifluorfen) to the diet resulted in a similar pattern of liver porphyrin accumulation in male ICR mice after three months of treatment. Uroporphyrin I was found to predominate over uroporphyrin III in both fomesafen and Blazer-treated groups.

Table I V . The Effect of Fomesafen and Iron Treatment on Liver and Kidney Porphyrin Concentrations of Male I C R Mice Treatment

Liver Porphyrin Content (nmol/g)

Control Fomesafen Fomesafeniron

1.0 + 0.5 138.2 ± 152.3 190.1 ± 55.8

Kidney Porphyrin Content (nmol/g) 0.7 + 0.1 3.5 + 2.1 12.0 + 3.0

Bile Porphyrin Content (nmol/g) 9.3+ 5.3 186.6 + 103.8 473.2 ± 144.9

Mice were fed 0.25% of fomesafen in the diet for 5 months,iron pretreated groups received 600mg/kg of iron by subcutaneous injection (n=4).

It has been demonstrated many times that the experimental porphyrias induced by polyhalogenated aromatic hydrocarbons, as well as the human disease porphyria cutanea tarda, can be modulated by manipulating the body iron stores. Iron pretreatment accelerates the development of hexachlorobenzene-induced uroporphyria, while phlebotomy is a widely used therapeutic approach to porphyria cutanea tarda (3). As can be seen in Table IV, iron pretreatment further significantly increased the elevated porphyrin levels induced by fomesafen. Thus, the mechanism of uroporphyria induction by fomesafen and polychlorinated aromatic hydrocarbons appears to be to some extent similar. Modulation of Hepatic Microsomal Cytochrome P450 Activities by Diphenyl Ether Herbicides. Microsomal cytochromes of the P450 family are the major hepatic hemeproteins. Their levels and activities can be altered by drug pretreatment and they play a key role in most experimental porphyrias, as well as in detoxification and/or activation of many xenobiotics. Typically, all porphyrogenic aromatic hydrocarbons induce cytochromes of the P450IA family in susceptible mouse strains (8). As can be seen in Table V , the P450IA-related ethoxyresorufin O-deethylase (EROD) activity was not induced by fomesafen or oxyfluorfen treatment in


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C57B1/6J mice, although this mouse strain is highly susceptible to P450IA induction by porphyrogenic aromatic hydrocarbons. Oxyfluorfen caused a moderate increase in hepatic pentoxyresorufin O-dealkylase (PROD) activity, which is typically induced by the model drug phénobarbital.

Table V . Changes in Cytochrome P450 Content and Activities after Long Term Treatment with Diphenyl Ether Herbicides Treatment

Total Microsomal P450 (nmol/mg)

EROD (pmol/mg/min)

PROD (pmol/mg/min)

Control Oxyfluorfen Fomesafen

0.60 ± 0 . 0 9 0.41 ± 0.02 0.64 ± 0.05

75 ± 13 73 ± 13 75 ± 9

18 ± 2 61 ± 10 30 ± 2

Male C57B1/6J mice were fed 2500 ppm of the herbicides in the diet for 3 months. Results are expressed per mg of microsomal protein.

The inability of diphenyl ethers to induce hepatic EROD activity indicates that the development of PCT-type porphyria after fomesafen treatment is not caused by dibenzodioxin or dibenzofuran contamination of the herbicide preparations. The Effect of Protoporphyrinogen Oxidase-inhibiting Herbicides on Porphyrin Accumulation in Human Hepatoma-derived HepG2 Cells. Diphenyl ethers and oxadiazon have been reported to increase the porphyrin content of rat hepatocytes in culture (9JO). Figure 1 shows the effect of three photobleaching herbicides on the cellular porphyrin content of human hepatomaderived HepG2 cells. It is evident that all three herbicides increased the cellular porphyrin content over a wide concentration range. Although oxyfluorfen and oxadiazon were more potent than fomesafen in the in vivo experiments, the extent of in vitro porphyrin accumulation is roughly similar for all three herbicides. This probably reflects in vivo differences in absorption and metabolism of these compounds, or different interactions with hepatic cytochromes P450. Toxicological Aspects of the Protoporphyrinogen Herbicides in Experimental Animals and M a n .

Oxidase-inhibiting

The presented results clearly indicate that the protoporphyrinogen oxidaseinhibiting herbicides can influence the porphyrin and heme metabolism in mammals. Although very high doses (2500 or 5000 ppm) were used in most experiments, it is evident that fomesafen has a significant effect on porphyrin accumulation at much lower daily doses of circa 20 mg/kg. In addition, significantly increased liver porphyrin concentrations were reported after feeding of 50 ppm of oxadiazon to male ICR mice for 14 days (77).


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0.000

0.004 0.063 Herbicide concentration (mM)

0.250

Figure 1. The effect of fomesafen (horizontally hatched bars), oxyfluorfen (diagonally hatched bars), and oxadiazon (closed bars) on the porphyrin content of HepG2 cells in culture. Cells were incubated with the herbicides for 24 hours, porphyrins were determined in washed cells after extraction by methanol/sulphuric acid.


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Previously published results show that the protoporphyrinogen oxidaseinhibiting herbicides can induce porphyrin accumulation in a variety of cells, including plant cells, rat hepatocytes, and human-derived HepG2 hepatoma cells. Consequently, these herbicides could theoretically interfere with the porphyrin biosynthetic pathway in man as well. However, rather high doses are needed to produce relatively slight changes in porphyrin concentrations in vivo. Also, the accumulation of liver porphyrins induced by fomesafen treatment is clearly reversible (Table III). Therefore, it is perhaps reasonable to speculate that, in view of these results, the risk of a serious disturbance of heme metabolism caused by exposure to protoporphyrinogen oxidase-inhibiting herbicides is small. An inherited enzymatic defect of the heme biosynthetic pathway is found in human porphyrias. Protoporphyrinogen oxidase deficiency is characteristic of variegate porphyria. Although acute attacks of variegate porphyria can be lifethreatening, a proportion of subjects with impaired activity of protoporphyrinogen oxidase can remain asymptomatic throughout life (72). This fact further supports the assumption that a slight, transient decrease in protoporphyrinogen oxidase activity - i f it occurred - would have no serious consequences. Prolonged treatment with high doses of fomesafen or acifluorfen resulted in an unexpected accumulation of highly carboxylated porphyrins in the liver of ICR mice. Accumulation of uroporphyrin has also been reported in in vitro experiments with acifluorfen-methyl (9). The observed pattern of porphyrin accumulation and excretion in vivo is a typical diagnostic feature of human porphyria cutanea tarda. The same porphyrin pattern is also encountered in acquired porphyrias induced by polychlorinated aromatic hydrocarbons, both in experimental animals and man. Although this type of porphyria only developed when extremely high doses of fomesafen or acifluorfen were administered for long periods, it is theoretically possible that these diphenyl ethers could potentiate the toxic effect of the known environmental pollutants like hexachlorobenzene, polychlorinated biphenyls, or T C D D . In this respect, it is also interesting to note that nearly all porphyrogenic aromatic hydrocarbons are carcinogenic, and that acifluorfen has also been classified as a probable human carcinogen (75). Notwithstanding the fact that the porphyrogenic action of diphenyl ethers and polychlorinated aromatic hydrocarbons is to some extent similar, their interaction seems to have little possible toxicological consequence for man. As pointed out, only extremely high doses of the diphenyl ethers induce a porphyria cutanea tardalike porphyria in rodents (fomesafen is about two orders of magnitude less potent than hexachlorobenzene when the total porphyrogenic doses are compared). The porphyrogenic polychlorinated aromatic hydrocarbons are persistent environmental pollutants and their levels are carefully monitored, which further reduces the risk of interaction. Also, the available data on diphenyl ethers point to a relatively rapid excretion (14), as compared to the polyhalogenated aromatic hydrocarbons. Thus, it can be again concluded that the diphenyl ethers would probably pose a small toxicological hazard to man when properly used. However, since the heme moiety, of which protoporphyrinogen is a precursor, has a wide variety of specific functions, further experiments are needed to definitely confirm this assumption.


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Acknowledgments This work was supported by Charles University Grant No.268. A significant part of the experiments was generously funded and supported by the Rotary Club Baarn-Soest, the Netherlands.

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Porphyric Pesticides - American Chemical Society

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